Temporal proteomics of human cerebrospinal fluid after severe traumatic brain injury.

The pathophysiology of traumatic brain injury (TBI) requires further characterization to fully elucidate changes in molecular pathways. Cerebrospinal fluid (CSF) provides a rich repository of brain-associated proteins. In this retrospective observational study, we implemented high-resolution mass spectrometry to evaluate changes to the CSF proteome after severe TBI. 91 CSF samples were analyzed with mass spectrometry, collected from 16 patients with severe TBI (mean 32 yrs; 81% male) on day 0, 1, 2, 4, 7 and/or 10 post-injury (8-16 samples/timepoint) and compared to CSF obtained from 11 non-injured controls. We quantified 1152 proteins with mass spectrometry, of which approximately 80% were associated with CSF. 1083 proteins were differentially regulated after TBI compared to control samples. The most highly-upregulated proteins at each timepoint included neutrophil elastase, myeloperoxidase, cathepsin G, matrix metalloproteinase-8, and S100 calcium-binding proteins A8, A9 and A12-all proteins involved in neutrophil activation, recruitment, and degranulation. Pathway enrichment analysis confirmed the robust upregulation of proteins associated with innate immune responses. Conversely, downregulated pathways included those involved in nervous system development, and several proteins not previously identified after TBI such as testican-1 and latrophilin-1. We also identified 7 proteins (GM2A, Calsyntenin 1, FAT2, GANAB, Lumican, NPTX1, SFRP2) positively associated with an unfavorable outcome at 6 months post-injury. Together, these findings highlight the robust innate immune response that occurs after severe TBI, supporting future studies to target neutrophil-related processes. In addition, the novel proteins we identified to be differentially regulated by severe TBI warrant further investigation as potential biomarkers of brain damage or therapeutic targets.

Dual-modality NIRF-MRI cubosomes and hexosomes: High throughput formulation and in vivo biodistribution.

Engineered nanoparticles with multiple complementary imaging modalities are of great benefit to the rapid treatment and diagnosis of disease in various organs. Herein, we report the formulation of cubosomes and hexosomes that carry multiple amphiphilic imaging contrast agents in their self-assembled lipid bilayers. This is the first report of the use of both near infrared fluorescent (NIRF) imaging and gadolinium lipid based magnetic resonance (MR) imaging modalities in cubosomes and hexosomes. High-throughput screening was used to rapidly optimize formulations with desirable nano-architectures and low in vitro cytotoxicity. The dual-modal imaging nanoparticles in vivo biodistribution and organ specific contrast enhancement were then studied. The NIRF in vivo imaging results indicated accumulation of both cubosomes and hexosomes in the liver and spleen of mice up to 20h post-injection. Remarkably, the biodistribution of the nanoparticle formulations was affected by the mesophase (i.e. cubic or hexagonal), a finding of significant importance for the future use of these compounds, with hexosomes showing higher accumulation in the spleen than the liver compared to cubosomes. Furthermore, in vivo MRI data of animals injected with either type of lyotropic liquid crystal nanoparticle displayed enhanced contrast in the liver and spleen.

Impact Acceleration Model of Diffuse Traumatic Brain Injury.

The impact acceleration (I/A) model of traumatic brain injury (TBI) was developed to reliably induce diffuse traumatic axonal injury in rats in the absence of skull fractures and parenchymal focal lesions. This model replicates a pathophysiology that is commonly observed in humans with diffuse axonal injury (DAI) caused by acceleration-deceleration forces. Such injuries are typical consequences of motor vehicle accidents and falls, which do not necessarily require a direct impact to the closed skull. There are several desirable characteristics of the I/A model, including the extensive axonal injury produced in the absence of a focal contusion, the suitability for secondary insult modeling, and the adaptability for mild/moderate injury through alteration of height and/or weight. Furthermore, the trauma device is inexpensive and readily manufactured in any laboratory, and the induction of injury is rapid (~45 min per animal from weighing to post-injury recovery) allowing multiple animal experiments per day. In this chapter, we describe in detail the methodology and materials required to produce the rat model of I/A in the laboratory. We also review current adaptations to the model to alter injury severity, discuss frequent complications and technical issues encountered using this model, and provide recommendations to ensure technically sound injury induction.

Erythropoietin improves motor and cognitive deficit, axonal pathology, and neuroinflammation in a combined model of diffuse traumatic brain injury and hypoxia, in association with upregulation of the erythropoietin receptor.

BACKGROUND: Diffuse axonal injury is a common consequence of traumatic brain injury (TBI) and often co-occurs with hypoxia, resulting in poor neurological outcome for which there is no current therapy. Here, we investigate the ability of the multifunctional compound erythropoietin (EPO) to provide neuroprotection when administered to rats after diffuse TBI alone or with post-traumatic hypoxia. METHODS: Sprague-Dawley rats were subjected to diffuse traumatic axonal injury (TAI) followed by 30 minutes of hypoxic (Hx, 12% O2) or normoxic ventilation, and were administered recombinant human EPO-α (5000 IU/kg) or saline at 1 and 24 hours post-injury. The parameters examined included: 1) behavioural and cognitive deficit using the Rotarod, open field and novel object recognition tests; 2) axonal pathology (NF-200); 3) callosal degradation (hematoxylin and eosin stain); 3) dendritic loss (MAP2); 4) expression and localisation of the EPO receptor (EpoR); 5) activation/infiltration of microglia/macrophages (CD68) and production of IL-1ß. RESULTS: EPO significantly improved sensorimotor and cognitive recovery when administered to TAI rats with hypoxia (TAI + Hx). A single dose of EPO at 1 hour reduced axonal damage in the white matter of TAI + Hx rats at 1 day by 60% compared to vehicle. MAP2 was decreased in the lateral septal nucleus of TAI + Hx rats; however, EPO prevented this loss, and maintained MAP2 density over time. EPO administration elicited an early enhanced expression of EpoR 1 day after TAI + Hx compared with a 7-day peak in vehicle controls. Furthermore, EPO reduced IL-1ß to sham levels 2 hours after TAI + Hx, concomitant to a decrease in CD68 positive cells at 7 and 14 days. CONCLUSIONS: When administered EPO, TAI + Hx rats had improved behavioural and cognitive performance, attenuated white matter damage, resolution of neuronal damage spanning from the axon to the dendrite, and suppressed neuroinflammation, alongside enhanced expression of EpoR. These data provide compelling evidence of EPO's neuroprotective capability. Few benefits were observed when EPO was administered to TAI rats without hypoxia, indicating that EPO's neuroprotective capacity is bolstered under hypoxic conditions, which may be an important consideration when EPO is employed for neuroprotection in the clinic.

Characterising effects of impact velocity on brain and behaviour in a model of diffuse traumatic axonal injury.

The velocity of impact between an object and the human head is a critical factor influencing brain injury outcomes but has not been explored in any detail in animal models. Here we provide a comprehensive overview of the interplay between impact velocity and injury severity in a well-established weight-drop impact acceleration (WDIA) model of diffuse brain injury in rodents. We modified the standard WDIA model to produce impact velocities of 5.4, 5.85 and 6.15 m/s while keeping constant the weight and the drop height. Gradations in impact velocity produced progressive degrees of injury severity measured behaviourally, electrophysiologically and anatomically, with the former two methods showing greater sensitivity to changes in impact velocity. There were impact velocity-dependent reductions in sensorimotor performance and in cortical depth-related depression of sensory cortex responses; however axonal injury (demonstrated by immunohistochemistry for ß-amyloid precursor protein and neurofilament heavy-chain) was discernible only at the highest impact velocity. We conclude that the WDIA model is capable of producing graded axonal injury in a repeatable manner, and as such will prove useful in the study of the biomechanics, pathophysiology and potential treatment of diffuse axonal injury.

Early management of severe traumatic brain injury.

Severe traumatic brain injury remains a major health-care problem worldwide. Although major progress has been made in understanding of the pathophysiology of this injury, this has not yet led to substantial improvements in outcome. In this report, we address present knowledge and its limitations, research innovations, and clinical implications. Improved outcomes for patients with severe traumatic brain injury could result from progress in pharmacological and other treatments, neural repair and regeneration, optimisation of surgical indications and techniques, and combination and individually targeted treatments. Expanded classification of traumatic brain injury and innovations in research design will underpin these advances. We are optimistic that further gains in outcome for patients with severe traumatic brain injury will be achieved in the next decade.

Inflammatory regulators of redirected neural migration in the injured brain.

Brain injury following stroke or trauma induces the migration of neuroblasts derived from subventricular zone neural precursor cells (NPCs) towards the damaged tissue, where they then have the potential to contribute to repair. Enhancing the recruitment of new cells thus presents an enticing prospect for the development of new therapeutic approaches to treat brain injury; to this end, an understanding of the factors regulating this process is required. During the neuroinflammatory response to ischemic and traumatic brain injuries, a plethora of pro- and anti-inflammatory cytokines, chemokines and growth factors are released in the damaged tissue, and recent work indicates that a variety of these are able to influence injury-induced migration. In this review, we will discuss the contribution of specific chemokines and growth factors towards stimulating NPC migration in the injured brain.

Attenuation of microglial activation with minocycline is not associated with changes in neurogenesis after focal traumatic brain injury in adult mice.

Neurogenesis is stimulated following injury to the adult brain and could potentially contribute to tissue repair. However, evidence suggests that microglia activated in response to injury are detrimental to the survival of new neurons, thus limiting the neurogenic response. The aim of this study was to determine the effect of the anti-inflammatory drug minocycline on neurogenesis and functional recovery after a closed head injury model of focal traumatic brain injury (TBI). Beginning 30 min after trauma, minocycline was administered for up to 2 weeks and bromodeoxyuridine was given on days 1-4 to label proliferating cells. Neurological outcome and motor function were evaluated over 6 weeks using the Neurological Severity Score (NSS) and ledged beam task. Microglial activation was assessed in the pericontusional cortex and hippocampus at 1 week post-trauma, using immunohistochemistry to detect F4/80. Following immunolabeling of bromodeoxyuridine, double-cortin, and NeuN, cells undergoing distinct stages of neurogenesis, including proliferation, neuronal differentiation, neuroblast migration, and long-term survival, were quantified at 1 and 6 weeks in the hippocampal dentate gyrus, as well as in the subventricular zone of the lateral ventricles and the pericontusional cortex. Our results show that minocycline successfully reduced microglial activation and promoted early neurological recovery that was sustained over 6 weeks. We also show for the first time in the closed head injury model, that early stages of neurogenesis were stimulated in the hippocampus and subventricular zone; however, no increase in new mature neurons occurred. Contrary to our hypothesis, despite the attenuation of activated microglia, minocycline did not support neurogenesis in the hippocampus, lateral ventricles, or pericontusional cortex, with none of the neurogenic stages being affected by treatment. These data provide evidence that a general suppression of microglial activation is insufficient to enhance neuronal production, suggesting that further work is required to elucidate the relationship between microglia and neurogenesis after TBI.

Post-traumatic hypoxia exacerbates neurological deficit, neuroinflammation and cerebral metabolism in rats with diffuse traumatic brain injury.

BACKGROUND: The combination of diffuse brain injury with a hypoxic insult is associated with poor outcomes in patients with traumatic brain injury. In this study, we investigated the impact of post-traumatic hypoxia in amplifying secondary brain damage using a rat model of diffuse traumatic axonal injury (TAI). Rats were examined for behavioral and sensorimotor deficits, increased brain production of inflammatory cytokines, formation of cerebral edema, changes in brain metabolism and enlargement of the lateral ventricles. METHODS: Adult male Sprague-Dawley rats were subjected to diffuse TAI using the Marmarou impact-acceleration model. Subsequently, rats underwent a 30-minute period of hypoxic (12% O2/88% N2) or normoxic (22% O2/78% N2) ventilation. Hypoxia-only and sham surgery groups (without TAI) received 30 minutes of hypoxic or normoxic ventilation, respectively. The parameters examined included: 1) behavioural and sensorimotor deficit using the Rotarod, beam walk and adhesive tape removal tests, and voluntary open field exploration behavior; 2) formation of cerebral edema by the wet-dry tissue weight ratio method; 3) enlargement of the lateral ventricles; 4) production of inflammatory cytokines; and 5) real-time brain metabolite changes as assessed by microdialysis technique. RESULTS: TAI rats showed significant deficits in sensorimotor function, and developed substantial edema and ventricular enlargement when compared to shams. The additional hypoxic insult significantly exacerbated behavioural deficits and the cortical production of the pro-inflammatory cytokines IL-6, IL-1ß and TNF but did not further enhance edema. TAI and particularly TAI+Hx rats experienced a substantial metabolic depression with respect to glucose, lactate, and glutamate levels. CONCLUSION: Altogether, aggravated behavioural deficits observed in rats with diffuse TAI combined with hypoxia may be induced by enhanced neuroinflammation, and a prolonged period of metabolic dysfunction.

Neurogenesis and glial proliferation are stimulated following diffuse traumatic brain injury in adult rats.

Although increased neurogenesis has been described in rodent models of focal traumatic brain injury (TBI), the neurogenic response occurring after diffuse TBI uncomplicated by focal injury has not been examined to date, despite the pervasiveness of this distinct type of brain injury in the TBI patient population. Here we characterize multiple stages of neurogenesis following a traumatic axonal injury (TAI) model of diffuse TBI as well as the proliferative response of glial cells. TAI was induced in adult rats using an impact-acceleration model, and 5-bromo-2'-deoxyuridine (BrdU) was administered on days 1-4 posttrauma or sham operation to label mitotic cells. Using immunohistochemistry for BrdU combined with phenotype-specific markers, we found that proliferation was increased following TAI in the subventricular zone of the lateral ventricles and in the hippocampal subgranular zone, although the ultimate production of new dentate granule neurons at 8 weeks was not significantly enhanced. Also, abundant proliferating and reactive astrocytes, microglia, and polydendrocytes were detected throughout the brain following TAI, indicating that a robust glial response occurs in this model, although very few new cells in the nonneurogenic brain regions became mature neurons. We conclude that diffuse brain injury stimulates early stages of a neurogenic response similar to that described for models of focal TBI.

CCL2 modulates cytokine production in cultured mouse astrocytes.

BACKGROUND: The chemokine CCL2 (also known as monocyte chemoattractant protein-1, or MCP-1) is upregulated in patients and rodent models of traumatic brain injury (TBI), contributing to post-traumatic neuroinflammation and degeneration by directing the infiltration of blood-derived macrophages into the injured brain. Our laboratory has previously reported that Ccl2-/- mice show reduced macrophage accumulation and tissue damage, corresponding to improved motor recovery, following experimental TBI. Surprisingly, Ccl2-deficient mice also exhibited delayed but exacerbated secretion of key proinflammatory cytokines in the injured cortex. Thus we sought to further characterise CCL2's potential ability to modulate immunoactivation of astrocytes in vitro. METHODS: Primary astrocytes were isolated from neonatal wild-type and Ccl2-deficient mice. Established astrocyte cultures were stimulated with various concentrations of lipopolysaccharide (LPS) and interleukin (IL)-1ß for up to 24 hours. Separate experiments involved pre-incubation with mouse recombinant (r)CCL2 prior to IL-1ß stimulation in wild-type cells. Following stimulation, cytokine secretion was measured in culture supernatant by immunoassays, whilst cytokine gene expression was quantified by real-time reverse transcriptase polymerase chain reaction. RESULTS: LPS (0.1-100 µg/ml; 8 h) induced the significantly greater secretion of five key cytokines and chemokines in Ccl2-/- astrocytes compared to wild-type cells. Consistently, IL-6 mRNA levels were 2-fold higher in Ccl2-deficient cells. IL-1ß (10 and 50 ng/ml; 2-24 h) also resulted in exacerbated IL-6 production from Ccl2-/- cultures. Despite this, treatment of wild-type cultures with rCCL2 alone (50-500 ng/ml) did not induce cytokine/chemokine production by astrocytes. However, pre-incubation of wild-type astrocytes with rCCL2 (250 ng/ml, 12 h) prior to stimulation with IL-1ß (10 ng/ml, 8 h) significantly reduced IL-6 protein and gene expression. CONCLUSIONS: Our data indicate that astrocytes are likely responsible for the exacerbated cytokine response seen in vivo post-injury in the absence of CCL2. Furthermore, evidence that CCL2 inhibits cytokine production by astrocytes following IL-1ß stimulation, suggests a novel, immunomodulatory role for this chemokine in acute neuroinflammation. Further investigation is required to determine the physiological relevance of this phenomenon, which may have implications for therapeutics targeting CCL2-mediated leukocyte infiltration following TBI.

Post-traumatic hypoxia exacerbates brain tissue damage: analysis of axonal injury and glial responses.

Traumatic brain injury (TBI) resulting in poor neurological outcome is predominantly associated with diffuse brain damage and secondary hypoxic insults. Post-traumatic hypoxia is known to exacerbate primary brain injury; however, the underlying pathological mechanisms require further elucidation. Using a rat model of diffuse traumatic axonal injury (TAI) followed by a post-traumatic hypoxic insult, we characterized axonal pathology, macrophage/microglia accumulation, and astrocyte responses over 14 days. Rats underwent TAI alone, TAI followed by 30 min of hypoxia (TAI + Hx), hypoxia alone, or sham-operation (n = 6/group). Systemic hypoxia was induced by ventilating rats with 12% oxygen in nitrogen, resulting in a ∼ 50% reduction in arterial blood oxygen saturation. Brains were assessed for axonal damage, macrophage/microglia accumulation, and astrocyte activation at 1, 7, and 14 days post-treatment. Immunohistochemistry with axonal damage markers (ß-amyloid precursor protein [ß-APP] and neurofilament) showed strong positive staining in TAI + Hx rats, which was most prominent in the corpus callosum (retraction bulbs 69.8 ± 18.67; swollen axons 14.2 ± 5.25), and brainstem (retraction bulbs 294 ± 118.3; swollen axons 50.3 ± 20.45) at 1 day post-injury. Extensive microglia/macrophage accumulation detected with the CD68 antibody was maximal at 14 days post-injury in the corpus callosum (macrophages 157.5 ± 55.48; microglia 72.71 ± 20.75), and coincided with regions of axonal damage. Astrocytosis assessed with glial fibrillary acidic protein (GFAP) antibody was also abundant in the corpus callosum and maximal at 14 days, with a trend toward an increase in TAI + Hx animals (18.99 ± 2.45 versus 13.56 ± 0.81; p = 0.0617). This study demonstrates for the first time that a hypoxic insult following TAI perpetuates axonal pathology and cellular inflammation, which may account for the poor neurological outcomes seen in TBI patients who experience post-traumatic hypoxia.

Deficiency of the chemokine receptor CXCR2 attenuates neutrophil infiltration and cortical damage following closed head injury.

The contribution of infiltrated neutrophils to secondary damage following traumatic brain injury remains controversial. Chemokines that regulate neutrophil migration by signaling through the CXCR2 receptor are markedly elevated by brain injury and are associated with the propagation of secondary damage. This study thus investigated the function of CXCR2 in posttraumatic inflammation and secondary degeneration by examining Cxcr2-deficient (Cxcr2(-/-)) mice over 14 days following closed head injury (CHI). We demonstrate a significant attenuation of neutrophil infiltration in Cxcr2(-/-) mice at 12 hours and 7 days after CHI, despite increased levels of CXC neutrophil-attracting chemokines in the lesioned cortex. This coincides with reduced tissue damage, neuronal loss, and cell death in Cxcr2(-/-) mice compared to wild-type controls, with heterozygotes showing intermediate responses. In contrast, blood-brain barrier permeability and functional recovery did not appear to be affected by Cxcr2 deletion. This study highlights the deleterious contribution of neutrophils to posttraumatic neurodegeneration and demonstrates the importance of CXC chemokine signaling in this process. Therefore, CXCR2 antagonistic therapeutics currently in development for other inflammatory conditions may also be of benefit in posttraumatic neuroinflammation.

Animal models of traumatic brain injury: is there an optimal model to reproduce human brain injury in the laboratory?

Compared to other neurological diseases, the research surrounding traumatic brain injury (TBI) has a more recent history. The establishment and use of animal models of TBI remains vital to understand the pathophysiology of this highly complex disease. Such models share the ultimate goals of reproducing patterns of tissue damage observed in humans (thus rendering them clinically relevant), reproducible and highly standardised to allow for the manipulation of individual variables, and to finally explore novel therapeutics for clinical translation. There is no doubt that the similarity of cellular and molecular events observed in human and rodent TBI has reinforced the use of small animals for research. When confronted with the choice of the experimental model it becomes clear that the ideal animal model does not exist. This limitation derives from the fact that most models mimic either focal or diffuse brain injury, whereas the clinical reality suggests that each patient has an individual form of TBI characterised by various combinations of focal and diffuse patterns of tissue damage. This is additionally complicated by the occurrence of secondary insults such as hypotension, hypoxia, ischaemia, extracranial injuries, modalities of traumatic events, age, gender and heterogeneity of medical treatments and pre-existing conditions. This brief review will describe the variety of TBI models available for laboratory research beginning from the most widely used rodent models of focal brain trauma, to complex large species such as the pig. In addition, the models mimicking diffuse brain damage will be discussed in relation to the early primate studies until the use of most common rodent models to elucidate the intriguing and less understood pathology of axonal dysfunction. The most recent establishment of in vitro paradigms has complemented the in vivo modelling studies offering a further cellular and molecular insight of this pathology.

Role of CCL2 (MCP-1) in traumatic brain injury (TBI): evidence from severe TBI patients and CCL2-/- mice.

Cerebral inflammation involves molecular cascades contributing to progressive damage after traumatic brain injury (TBI). The chemokine CC ligand-2 (CCL2) (formerly monocyte chemoattractant protein-1, MCP-1) is implicated in macrophage recruitment into damaged parenchyma after TBI. This study analyzed the presence of CCL2 in human TBI, and further investigated the role of CCL2 in physiological and cellular mechanisms of secondary brain damage after TBI. Sustained elevation of CCL2 was detected in the cerebrospinal fluid (CSF) of severe TBI patients for 10 days after trauma, and in cortical homogenates of C57Bl/6 mice, peaking at 4 to 12 h after closed head injury (CHI). Neurological outcome, lesion volume, macrophage/microglia infiltration, astrogliosis, and the cerebral cytokine network were thus examined in CCL2-deficient (-/-) mice subjected to CHI. We found that CCL2-/- mice showed altered production of multiple cytokines acutely (2 to 24 h); however, this did not affect lesion size or cell death within the first week after CHI. In contrast, by 2 and 4 weeks, a delayed reduction in lesion volume, macrophage accumulation, and astrogliosis were observed in the injured cortex and ipsilateral thalamus of CCL2-/- mice, corresponding to improved functional recovery as compared with wild-type mice after CHI. Our findings confirm the significant role of CCL2 in mediating post-traumatic secondary brain damage.

The complex and diversified mitochondrial gene pool of Berber populations.

The mitochondrial DNA variation of 295 Berber-speakers from Morocco (Asni, Bouhria and Figuig) and the Egyptian oasis of Siwa was evaluated by sequencing a portion of the control region (including HVS-I and part of HVS-II) and surveying haplogroup-specific coding region markers. Our findings show that the Berber mitochondrial pool is characterized by an overall high frequency of Western Eurasian haplogroups, a somehow lower frequency of sub-Saharan L lineages, and a significant (but differential) presence of North African haplogroups U6 and M1, thus occupying an intermediate position between European and sub-Saharan populations in PCA analysis. A clear and significant genetic differentiation between the Berbers from Maghreb and Egyptian Berbers was also observed. The first are related to European populations as shown by haplogroup H1 and V frequencies, whereas the latter share more affinities with East African and Nile Valley populations as indicated by the high frequency of M1 and the presence of L0a1, L3i, L4*, and L4b2 lineages. Moreover, haplogroup U6 was not observed in Siwa. We conclude that the origins and maternal diversity of Berber populations are old and complex, and these communities bear genetic characteristics resulting from various events of gene flow with surrounding and migrating populations.

Endogenous melatonin increases in cerebrospinal fluid of patients after severe traumatic brain injury and correlates with oxidative stress and metabolic disarray.

Oxidative stress plays a significant role in secondary damage after severe traumatic brain injury (TBI); and melatonin exhibits both direct and indirect antioxidant effects. Melatonin deficiency is deleterious in TBI animal models, and its administration confers neuroprotection, reducing cerebral oedema, and improving neurobehavioural outcome. This study aimed to measure the endogenous cerebrospinal fluid (CSF) and serum melatonin levels post-TBI in humans and to identify relationships with markers of oxidative stress via 8-isoprostaglandin-F2alpha (isoprostane), brain metabolism and neurologic outcome. Cerebrospinal fluid and serum samples of 39 TBI patients were assessed for melatonin, isoprostane, and various metabolites. Cerebrospinal fluid but not serum melatonin levels were markedly elevated (7.28+/-0.92 versus 1.47+/-0.35 pg/mL, P<0.0005). Isoprostane levels also increased in both CSF (127.62+/-16.85 versus 18.28+/-4.88 pg/mL, P<0.0005) and serum (562.46+/-50.78 versus 126.15+/-40.08 pg/mL (P<0.0005). A strong correlation between CSF melatonin and CSF isoprostane on day 1 after injury (r=0.563, P=0.002) suggests that melatonin production increases in conjunction with lipid peroxidation in TBI. Relationships between CSF melatonin and pyruvate (r=0.369, P=0.049) and glutamate (r=0.373, P=0.046) indicate that melatonin production increases with metabolic disarray. In conclusion, endogenous CSF melatonin levels increase after TBI, whereas serum levels do not. This elevation is likely to represent a response to oxidative stress and metabolic disarray, although further studies are required to elucidate these relationships.

Immunohistochemical characterization of Fas (CD95) and Fas Ligand (FasL/CD95L) expression in the injured brain: relationship with neuronal cell death and inflammatory mediators.

Traumatic brain injury causes progressive tissue atrophy and consequent neurological dysfunction, resulting from neuronal cell death in both animal models and patients. Fas (CD95) and Fas ligand (FasL/CD95L) are important mediators of apoptosis. However, little is known about the relationship between Fas and FasL and neuronal cell death in mice lacking the genes for inflammatory cytokines. In the present study, double tumor necrosis factor/lymphotoxin-alpha knockout (-/-) and interleukin-6-/- mice were subjected to closed head injury (CHI) and sacrificed at 24 hours or 7 days post-injury. Consecutive brain sections were evaluated for Fas and FasL expression, in situ DNA fragmentation (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling; TUNEL), morphologic characteristics of apoptotic cell death and leukocyte infiltration. A peak incidence of TUNEL positive cells was found in the injured cortex at 24 hours which remained slightly elevated at 7 days and coincided with maximum Fas expression. FasL was only moderately increased at 24 hours and showed maximum expression at 7 days. A few TUNEL positive cells were also found in the ipsilateral hippocampus at 24 hours. Apoptotic, TUNEL positive cells mostly co-localized with neurons and Fas and FasL immunoreactivity. The amount of accumulated polymorphonuclear leukocytes and CD11b positive cells was maximal in the injured hemispheres at 24 hours. We show strong evidence that Fas and FasL might be involved in neuronal apoptosis after CHI. Furthermore, Fas and FasL upregulation seems to be independent of neuroinflammation since no differences were found between cytokine-/- and wild-type mice.

Transient neuroprotection by minocycline following traumatic brain injury is associated with attenuated microglial activation but no changes in cell apoptosis or neutrophil infiltration.

Cerebral inflammation and apoptotic cell death are two processes implicated in the progressive tissue damage that occurs following traumatic brain injury (TBI), and strategies to inhibit one or both of these pathways are being investigated as potential therapies for TBI patients. The tetracycline derivative minocycline was therapeutically effective in various models of central nervous system injury and disease, via mechanisms involving suppression of inflammation and apoptosis. We therefore investigated the effect of minocycline in TBI using a closed head injury model. Following TBI, mice were treated with minocycline or vehicle, and the effect on neurological outcome, lesion volume, inflammation and apoptosis was evaluated for up to 7 days. Our results show that while minocycline decreases lesion volume and improves neurological outcome at 1 day post-trauma, this response is not maintained at 4 days. The early beneficial effect is likely not due to anti-apoptotic mechanisms, as the density of apoptotic cells is not affected at either time-point. However, protection by minocycline is associated with a selective anti-inflammatory response, in that microglial activation and interleukin-1beta expression are reduced, while neutrophil infiltration and expression of multiple cytokines are not affected. These findings demonstrate that further studies on minocycline in TBI are necessary in order to consider it as a novel therapy for brain-injured patients.

Cytomegalovirus production by infected astrocytes correlates with transforming growth factor-beta release.

Cytomegalovirus (CMV) encephalitis is well documented in immunosuppressed persons, but its pathogenesis has received little investigative attention. The examination of brain tissue from 2 patients with acquired immunodeficiency syndrome who had CMV encephalitis showed colocalization of CMV inclusions and transforming growth factor (TGF)-beta in cells that contained astrocyte-specific glial filaments. To investigate the relationship between CMV and TGF-beta in the brain, an ex vivo murine model of CMV-infected astrocytes was established. Cultures of primary murine (strain FVB/N) astrocytes inoculated with murine (Smith strain) CMV expressed, over time, increasing amounts of infectious CMV in parallel with increasing levels of TGF-beta mRNA and peptide. Astrocyte release of CMV declined in the presence of antibody to TGF-beta and increased substantially after the addition of exogenous TGF-beta. These findings suggest that CMV infection of astrocytes induces the production of TGF-beta, which in turn enhances productive CMV expression.