Plasma von Willebrand Factor Is Elevated Hyperacutely After Mild Traumatic Brain Injury.

Each year in the United States, ∼2.7 million persons seek medical attention for traumatic brain injury (TBI), of which ∼85% are characterized as being mild brain injuries. Many different cell types in the brain are affected in these heterogeneous injuries, including neurons, glia, and the brain vasculature. Efforts to identify biomarkers that reflect the injury of these different cell types have been a focus of ongoing investigation. We hypothesized that von Willebrand factor (vWF) is a sensitive biomarker for acute traumatic vascular injury and correlates with symptom severity post-TBI. To address this, blood was collected from professional boxing athletes (n = 17) before and within 30 min after competition. Plasma levels of vWF and neuron-specific enolase were measured using the Meso Scale Discovery, LLC. (MSD) electrochemiluminescence array-based multi-plex format (MSD, Gaithersburg, MD). Additional symptom and outcome data from boxers and patients, such as the Rivermead symptom scores (Rivermead Post Concussion Symptoms Questionnaire [RPQ-3]), were collected. We found that, subsequent to boxing bouts, there was a 1.8-fold increase in vWF levels within 30 min of injury (p < 0.0009). Moreover, fold-change in vWF correlates moderately (r = 0.51; p = 0.03) with the number of head blows. We also found a positive correlation (r = 0.69; p = 0.002) between fold-change in vWF and self-reported post-concussive symptoms, measured by the RPQ-3. The receiver operating curve analysis of vWF plasma levels and RPQ-3 scoring yielded a sensitivity of 94.12% and a specificity of 76.5% with an area under the curve of 83% for boxers after a fight compared to the pre-bout baseline. This study suggests that vWF is a potential blood biomarker measurable in the hyperacute period after blunt mild TBI. This biomarker may prove to be useful in diagnosing and monitoring traumatic vascular injury.

Tau Is Elevated in Pediatric Patients on Extracorporeal Membrane Oxygenation.

Neurologic injury is a known and feared complication of extracorporeal membrane oxygenation (ECMO). Neurologic biomarkers may have a role in assisting in early identification of such. Axonal biomarker tau has not been investigated in the pediatric ECMO population. The objective of this study is to evaluate plasma levels of tau in pediatric patients supported with ECMO. Eighteen patients requiring ECMO support in a quaternary pediatric intensive care unit at a university-affiliated children's hospital from October 2015 to February 2017 were enrolled. Patients undergoing extracorporeal cardiopulmonary resuscitation or recent history of bypass were excluded. Plasma tau was measured using enzyme-linked immunosorbent assay. Neuroimaging was reviewed for acute neurologic injury, and tau levels were analyzed to assess for correlation. Tau was significantly higher in ECMO patients than in control subjects. Sixty-one percent of subjects had evidence of acute brain injury on neuroimaging, but tau level did not correlate with injury. Subjects with multifocal injury all experienced infarction and had significantly higher tau levels on ECMO day 3 than patients with isolated injury. In addition, peak tau levels of neuro-injured subjects were compared with controls and noninjured ECMO subjects using receiver operating curve analysis. This study demonstrates preliminary evidence of axonal injury in pediatric ECMO patients.

Plasma creatine kinase B correlates with injury severity and symptoms in professional boxers.

INTRODUCTION: Each year in the United States, approximately 1.7 million people sustain a traumatic brain injury (TBI). Of these TBI events, about 75 percent are characterized as being mild brain injuries. Immediately following TBI, a secondary brain damage persists for hours, days, and even months. Previously, detection of neuronal and glial biomarkers have proven to be useful to predict neurological outcomes. Here, we hypothesized that creatine kinase, brain (CKBB) is a sensitive biomarker for acute secondary brain injury in professional boxers. METHODS: Blood (8cc) was collected from the boxing athletes (n=18) prior to and after competition (∼30min). The plasma levels of CKBB were measured using the Meso Scale Diagnostic (MSD) electrochemiluminescence (ECL) array-based multiplex format. Additional data such as number of blows to the head and symptom score (Rivermead Post Concussion Symptoms Questionnaire) were collected. RESULTS: At approximately 30min after the competition, the plasma levels of CKBB were significantly elevated in concussed professional boxers and correlated with the number of blows to the head and symptom scores. Additionally, receiver operating curve (ROC) analysis yielded a 77.8% sensitivity and a specificity of 82.4% with an area under the curve (AUC) of 90% for CKBB as an identifier of secondary brain injury within this population. CONCLUSION: This study describes the detection of CKBB as a brain biomarker to detect secondary brain injury in professional athletes that have experienced multiple high impact blows to the head. This acute biomarker may prove useful in monitoring secondary brain injury after injury.

Adult obese mice suffer from chronic secondary brain injury after mild TBI.

BACKGROUND: A traumatic brain injury (TBI) event is a devastating injury to the brain that may result in heightened inflammation, neurodegeneration, and subsequent cognitive and mood deficits. TBI victims with co-morbidities such as heart disease, diabetes, or obesity may be more vulnerable to the secondary brain injury that follows the initial insult. Compared to lean individuals, obese subjects tend to have worse clinical outcomes and higher mortality rates after trauma. METHODS: To elucidate whether obesity predisposes individuals to worse outcomes after TBI, we subjected adult lean and obese male/female mice to a mild TBI. The injury was administered using a controlled skull impact (CSI) device. Lean or obese 6-month-old C57 BL/6 mice were subjected once to a mild TBI. Additionally, at day 30 after injury, both the lean and obese mice were tested for increased anxiety using the open field test. RESULTS: At day 30 after TBI, compared to the lean mice, we found heightened microglial (MG) activation in the cerebral cortex, corpus callosum, and hypothalamus. Another compelling finding was that, compared to the non-injured obese male control mice, the obese TBI mice had a decrease in the rate of weight gain and serum corticosterone levels at day 30 after injury. Additionally, the injured obese mice displayed higher levels of anxiety as determined by a significant decrease in time spent in the non-peripheral zones in the open field test. In contrast to the obese males, the obese female mice did not exhibit increases in the number of active MG in the brain, changes in weight gain/corticosterone levels, or increased anxiety at day 30 after TBI. CONCLUSIONS: The data presented here suggests that obese mice have worse outcomes compared to lean mice after mild TBI. Also, the obese males have worse outcomes than the injured female mice. This data may explain the sequela of chronic secondary brain injury in obese adults after a single mild TBI. Also, this report may help shape how the overweight/obese populations are monitored over the days and months following a TBI.

Evidence of increased brain amyloid in severe TBI survivors at 1, 12, and 24 months after injury: report of 2 cases.

Traumatic brain injury (TBI) is a major risk factor for Alzheimer's disease. With respect to amyloid deposition, there are no published serial data regarding the deposition rate of amyloid throughout the brain after TBI. The authors conducted serial (18)F-AV-45 (florbetapir F18) positron emission tomography (PET) imaging in 2 patients with severe TBI at 1, 12, and 24 months after injury. A total of 12 brain regions were surveyed for changes in amyloid levels. Case 1 involved a 50-year-old man who experienced a severe TBI. Compared with the 1-month time point, of the 12 brain regions that were surveyed, a decrease in amyloid (as indicated by standard uptake value ratios) was only observed in the hippocampus (-16%, left; -12%, right) and caudate nucleus (-18%, left; -18%, right), suggesting that initial amyloid accumulation in the brain was cleared between time points 1 and 12 months after injury. Compared to the scan at 1 year, a greater increase in amyloid (+15%) was observed in the right hippocampus at the 24-month time point. The patient in Case 2 was a 37-year-old man who suffered severe trauma to the head and a subsequent stroke; he had poor cognitive/functional outcomes and underwent 1.5 years of rehabilitation. Due to a large infarct area on the injured side of the brain (right side), the authors focused primarily on brain regions affected within the left hemisphere. Compared with the 1-month scan, they only found an increase in brain amyloid within the left anterior putamen (+11%) at 12 months after injury. In contrast, decreased amyloid burden was detected in the left caudate nucleus (-48%), occipital cortex (-21%), and precuneus (-19%) brain regions at the 12-month time point, which is indicative of early accumulation and subsequent clearance. In comparison with 12-month values, more clearance was observed, since a reduction in amyloid was found at 24 months after trauma within the left anterior putamen (-12%) and occipital cortex (-15%). Also, by 24 months, most of the amyloid had been cleared and the patient demonstrated improved results on the Rivermead symptom questionnaire, Glasgow Outcome Scale-Extended, and Disability Rating Scale. With respect to APOE status, the patient in Case 1 had two ε3 alleles and the patient in Case 2 had one ε2 and one ε3 allele. In comparison to the findings of the initial scan at 1 month after TBI, by 12 and 24 months after injury amyloid was cleared in some brain regions and increased in others. Serial imaging conducted here suggests that florbetapir F18 PET imaging may be useful in monitoring amyloid dynamics within specific brain regions following severe TBI and may be predictive of cognitive deficits.

High therapeutic potential of positive allosteric modulation of α7 nAChRs in a rat model of traumatic brain injury: proof-of-concept.

There are currently no clinically efficacious drug therapies to treat brain damage secondary to traumatic brain injury (TBI). In this proof-of-concept study, we used a controlled cortical impact model of TBI in young adult rats to explore a novel promising approach that utilizes PNU-120596, a previously reported highly selective Type-II positive allosteric modulator (α7-PAM) of α7 nicotinic acetylcholine receptors (nAChRs). α7-PAMs enhance and prolong α7 nAChR activation, but do not activate α7 nAChRs when administered without an agonist. The rational basis for the use of an α7-PAM as a post-TBI treatment is tripartite and arises from: (1) the intrinsic ability of brain injury to elevate extracellular levels of choline (a ubiquitous cell membrane-building material and a selective endogenous agonist of α7 nAChRs) due to the breakdown of cell membranes near the site and time of injury; (2) the ubiquitous expression of functional α7 nAChRs in neuronal and glial/immune brain cells; and (3) the potent neuroprotective and anti-inflammatory effects of α7 nAChR activation. Therefore, both neuroprotective and anti-inflammatory effects can be achieved post-TBI by targeting only a single player (i.e., the α7 nAChR) using α7-PAMs to enhance the activation of α7 nAChRs by injury-elevated extracellular choline. Our data support this hypothesis and demonstrate that subcutaneous administration of PNU-120596 post-TBI in young adult rats significantly reduces both brain cell damage and reactive gliosis. Therefore, our results introduce post-TBI systemic administration of α7-PAMs as a promising therapeutic intervention that could significantly restrict brain injury post-TBI and facilitate recovery of TBI patients.

Serum Levels of Neurofilament-H are Elevated in Patients Suffering From Severe Burns.

In previous studies, after injury, burn patients experienced an increase in neuro-inflammation, edema, and neuronal cell death. As demonstrated in other brain injury models, fluid-based biomarkers such as phosphorylated neurofilament-H (pNFL-H) have been shown to correlate with injury severity. In this study the authors hypothesized that burn-injured patients have an increase in pNFL-H in the blood during the acute and chronic time-points after injury. In this prospective clinical study, blood (8 cc) was collected from burn patients (n = 36; TBSA 10-60%) at Parkland hospital, Dallas, Texas, on days 1, 7, and 14 after injury. The serum levels of pNFL-H were measured using the enzyme-linked immunoassay. Compared to noninjured controls, the burn patients exhibited a significant increase in the serum levels of pNFL-H on days 7 (P < .0001) and 14 (P < .0001) after burn injury. No significant increase was observed on day 1 (P < .07) after injury. A positive correlation between TBSA and pNFL-H levels was observed for day 14 (r = .55; P < .03). Additionally, using the receiver operating characteristic analysis, the authors determined the area under the curve was 98% for both day 7 and 14. In conclusion, this study describes the serum profile of pNFL-H in patients suffering from severe burns during the acute (day 1) and chronic (days 7 and 14) time-points. These results suggest that detection of pNFL-H may be useful in determining which individuals suffer from nerve cell degeneration after burn.

Detection of neurofilament-H in serum as a diagnostic tool to predict injury severity in patients who have suffered mild traumatic brain injury.

OBJECT: In previous studies of traumatic brain injury (TBI), neural biomarkers of injury correlate with injury severity and predict neurological outcome. The object of this paper was to characterize neurofilament-H (NFL-H) as a predictor of injury severity in patients who have suffered mild TBI (mTBI). Thus, the authors hypothesized that phosphorylated NFL-H (pNFL-H) levels are higher in mTBI patients than in healthy controls and identify which subjects experienced a more severe injury such as skull fractures, intracranial hemorrhaging, and/or contusions as detected by CT scans. METHODS: In this prospective clinical study, blood (8 ml) was collected from subjects (n = 34) suffering from mTBI (as defined by the American Congress of Rehabilitation and Glasgow Coma Scale scores between 13 and 15) at Parkland Hospital, Dallas, Texas, on Days 1 and 3 after injury). Additional clinical findings from the CT scans were also used to categorize the TBI patients into those with and those without clinical findings on the scans (CT+ and CTgroups, respectively). The serum levels of pNFL-H were measured using the enzyme-linked immunosorbent assay. RESULTS: Compared with healthy controls, the mTBI patients exhibited a significant increase in the serum levels of pNFL-H on Days 1 (p = 0.00001) and 3 (p = 0.0001) after TBI. An inverse correlation was observed between pNFL-H serum levels and Glasgow Coma Scale scores, which was significant. Additionally, using receiver operating characteristic curve analysis to compare the mTBI cases with controls to determine sensitivity and specificity, an area under the curve of 100% was achieved for both (p = 0.0001 for both). pNFL-H serum levels were only significantly higher on Day 1 in mTBI patients in the CT+ group (p < 0.008) compared with the CT- group. The area under the curve (82.5%) for the CT+ group versus the CT- group was significant (p = 0.021) with a sensitivity of 87.5% and a specificity of 70%, using a cutoff of 1071 pg/ml of pNFL-H in serum. CONCLUSIONS: This study describes the serum profile of pNFL-H in patients suffering from mTBI with and without CT findings on Days 1 and 3 after injury. These results suggest that detection of pNFL-H may be useful in determining which individuals require CT imaging to assess the severity of their injury.

Non-feminizing estrogens: a novel neuroprotective therapy.

While the conflict between basic science evidence for estrogen neuroprotection and the lack of effectiveness in clinical trials is only now being resolved, it is clear that strategies for estrogen neuroprotection that avoid activation of ERs have the potential for clinical application. Herein we review the evidence from both in vitro and in vivo studies that describe high potency neuroprotection with non-feminizing estrogens. We have characterized many of the essential chemical features of non-feminizing estrogens that eliminate or reduce ER binding while maintaining or enhancing neuroprotection. Additionally, we provide evidence that these non-feminizing estrogens have efficacy in protecting the brain from AD neuropathology and traumatic brain injury. In conclusion, it appears that the non-feminizing estrogen strategy for neuroprotection is a viable option to achieve the beneficial neuroprotective effects of estrogens while eliminating the toxic off-target effects of chronic estrogen administration.

Resveratrol decreases inflammation in the brain of mice with mild traumatic brain injury.

BACKGROUND: Following a mild traumatic brain injury (TBI) event, the secondary brain injury that persists after the initial blow to the head consists of excitotoxicity, decreased cerebral glucose levels, oxidant injury, mitochondrial dysfunction, inflammation, and neuronal cell death. To date, there are no effective interventions used at decreasing secondary brain injury after mild TBI. METHODS: In this study, male mice were treated with either placebo or resveratrol (100 mg/kg) at 5 minutes and 12 hours after mild TBI. The mice were injured using the controlled cortical impact device. In this closed-head model, a midline incision was made to access the skull and the impactor tip was aligned on the sagittal suture midway between the bregma and lambda sutures. The mice were injured at a depth of 2.0 mm, velocity of 4 m/s, and a delay time of 100 milliseconds. At 72 hours following injury, the animals were intracardially perfused with 0.9% saline followed by 10% phosphate-buffered formalin. The whole brain was removed, sliced, and stained for microglial activation (Iba1). In addition, using the enzyme-linked immunosorbent assay, tissue levels of interleukin 6 (IL-6) and IL-12 were measured in the cerebral cortex and hippocampus. RESULTS: In this study, we found that in the placebo treatment group, there was a significant increase in Iba1 staining in the brain. The levels of microglial activation was reduced by resveratrol in the cerebral cortex (p < 0.001), corpus callosum (p < 0.001), and dentate gyrus (p < 0.005) brain regions after mild TBI. In addition to Iba1, resveratrol decreased the brain levels of IL-6 (p < 0.0001) and IL-12 (p < 0.004), which were observed in the hippocampus of the placebo group. In our model, no increase of IL-6 or IL-12 was observed in the cerebral cortex following TBI. CONCLUSION: Resveratrol given acutely after TBI results in a decrease in neuroinflammation. These results suggest that resveratrol may be beneficial in reducing secondary brain injury after experiencing a mild TBI.

17ß-Estradiol reappropriates mass lost to the hypermetabolic state in thermally injured rats.

BACKGROUND: The hypermetabolic response to severe thermal injury is unlike any physiologic response seen in medicine. While some parallels can be drawn to shock and sepsis states, this response is typified by its intensity and duration. Our group has been interested in the myriad effects of estrogens after injury, specifically the ability of estrogens to reduce inflammatory responses. Given this, and the known link between severe inflammation and the hypermetabolic response, we examined the effects of a single dose of 17ß estradiol administered after a severe thermal injury in rats. METHODS: Twelve male Sprague-Dawley rats were subject to either a sham burn or a 40% total body surface area burn, followed by fluid resuscitation. Burned animals were divided into a vehicle and treatment group, with injections given 15 min after the injury. Animals were monitored for a period of 45 d, with markers of hypermetabolism (weight, fecal output, food intake, and serum insulin and glucose) measured daily. RESULTS: We identified a significant difference in daily measured weights between the burned groups. We observed a sparing of body mass during the acute phase lasting 2 wk after the injury and an improved recovery phase during the remainder of the study. Glucose and insulin levels during the first week of the study did not differ between the treatment groups. CONCLUSION: Estrogen may have a role in preserving body mass after severe thermal injury. Further studies are required to determine if this spared body mass composition.

Estrone is neuroprotective in rats after traumatic brain injury.

In various animal and human studies, early administration of 17ß-estradiol, a strong antioxidant, anti-inflammatory, and anti-apoptotic agent, significantly decreases the severity of injury in the brain associated with cell death. Estrone, the predominant estrogen in postmenopausal women, has been shown to be a promising neuroprotective agent. The overall goal of this project was to determine if estrone mitigates secondary injury following traumatic brain injury (TBI) in rats. Male rats were given either placebo (corn oil) or estrone (0.5 mg/kg) at 30 min after severe TBI. Using a controlled cortical impact device in rats that underwent a craniotomy, the right parietal cortex was injured using the impactor tip. Non-injured control and sham animals were also included. At 72 h following injury, the animals were perfused intracardially with 0.9% saline followed by 10% phosphate-buffered formalin. The whole brain was removed, sliced, and stained for TUNEL-positive cells. Estrone decreased cortical lesion volume (p<0.01) and neuronal injury (p<0.001), and it reduced cerebral cortical levels of TUNEL-positive staining (p<0.0001), and decreased numbers of TUNEL-positive cells in the corpus callosum (p<0.03). We assessed the levels of ß-amyloid in the injured animals and found that estrone significantly decreased the cortical levels of ß-amyloid after brain injury. Cortical levels of phospho-ERK1/2 were significantly (p<0.01) increased by estrone. This increase was associated with an increase in phospho-CREB levels (p<0.021), and brain-derived neurotrophic factor (BDNF) expression (p<0.0006). In conclusion, estrone given acutely after injury increases the signaling of protective pathways such as the ERK1/2 and BDNF pathways, decreases ischemic secondary injury, and decreases apoptotic-mediated cell death. These results suggest that estrone may afford protection to those suffering from TBI.

Investigation of the temperature stability of premarin intravenous using liquid chromatography-mass spectrometry.

Stability of Premarin(®)Intravenous was investigated in dry and reconstituted forms by monitoring major components in samples for a period of six months, using liquid chromatography-mass spectrometry. The components, largely comprising a series of estrogen and steroid hormone sulfates, were considered to be fairly stable (variation≤10%) for dry samples stored at room temperature and at 38°C (100°F) during the experimental time frame. However, significant variation, especially after 2 months of storage, was observed in reconstituted solutions. This variation was significantly larger for samples stored at elevated vs. room temperature. It was interesting to note that the concentration of equilenin sulfate increased over time, whereas that of other major components were seen to fluctuate and decrease. This phenomenon was partially explained by the conversion of equilin compounds into their corresponding equilenin forms, a phenomenon which was further investigated through a storage study with pure standard solutions and by tandem mass spectrometry.

Simultaneous quantification of four native estrogen hormones at trace levels in human cerebrospinal fluid using liquid chromatography-tandem mass spectrometry.

Estrogens are known to exhibit neuroprotective effects on the brain. Their importance in this regard and in others has been emphasized in many recent studies, which increases the need to develop reliable analytical methods for the measurement of estrogen hormones. A heart-cutting two-dimensional liquid chromatography separation method coupled with electrospray ionization-tandem mass spectrometry (ESI-MS/MS) has been developed for simultaneous measurement of four estrogens, including estriol (E3), estrone (E1), 17ß-estradiol (17ß-E2), and 17α-estradiol (17α-E2), in human cerebrospinal fluid (CSF). The method was based on liquid-liquid extraction and derivatization of estrogens with dansyl chloride to enhance the sensitivity of ESI-based detection in conjunction with tandem mass spectrometry. Dansylated estriol and estrone were separated in the first dimension by an amide-C18 column, while dansylated 17ß- and 17α-estradiol were resolved on the second dimension by two C18 columns (175 mm total length) connected in series. This is the first report of a method for simultaneous quantification of all four endogenous estrogen compounds in their dansylated form. The detection limits for E1, 17α-E2, 17ß-E2, and E3 were 19, 35, 26, and 61pg/mL, respectively. Due to matrix effects, validation and calibration was carried out in charcoal-stripped CSF. The precision and accuracy were more than 86% for the two E2 compounds and 79% for E1 and E3 while the extraction recovery ranged from 91% to 104%. The method was applied to measure estrogens obtained in a clinical setting, from the CSF of ischemic trauma patients. While 17ß-estradiol was present at a significant level in the CSF of some samples, other estrogens were present at lower levels or were undetectable.

Protein kinase C and rho activated coiled coil protein kinase 2 (ROCK2) modulate Alzheimer's APP metabolism and phosphorylation of the Vps10-domain protein, SorL1.

BACKGROUND: Generation of the amyloid ß (Aß) peptide of Alzheimer's disease (AD) is differentially regulated through the intracellular trafficking of the amyloid ß precursor protein (APP) within the secretory and endocytic pathways. Protein kinase C (PKC) and rho-activated coiled-coil kinases (ROCKs) are two "third messenger" signaling molecules that control the relative utilization of these two pathways. Several members of the Vps family of receptors (Vps35, SorL1, SorCS1) play important roles in post-trans-Golgi network (TGN) sorting and generation of APP derivatives, including Aß at the TGN, endosome and the plasma membrane. We now report that Vps10-domain proteins are candidate substrates for PKC and/or ROCK2 and act as phospho-state-sensitive physiological effectors for post-TGN sorting of APP and its derivatives. RESULTS: Analysis of the SorL1 cytoplasmic tail revealed multiple consensus sites for phosphorylation by protein kinases. SorL1 was subsequently identified as a phosphoprotein, based on sensitivity of its electrophoretic migration pattern to calf intestine alkaline phosphatase and on its reaction with anti-phospho-serine antibodies. Activation of PKC resulted in increased shedding of the ectodomains of both APP and SorL1, and this was paralleled by an apparent increase in the level of the phosphorylated form of SorL1. ROCK2, the neuronal isoform of another protein kinase, was found to form complexes with SorL1, and both ROCK2 inhibition and ROCK2 knockdown enhanced generation of both soluble APP and Aß. CONCLUSION: These results highlight the potential importance of SorL1 in elucidating phospho-state sensitive mechanisms in the regulation of metabolism of APP and Aß by PKC and ROCK2.

Estrogen treatment following severe burn injury reduces brain inflammation and apoptotic signaling.

BACKGROUND: Patients with severe burn injury experience a rapid elevation in multiple circulating pro-inflammatory cytokines, with the levels correlating with both injury severity and outcome. Accumulations of these cytokines in animal models have been observed in remote organs, however data are lacking regarding early brain cytokine levels following burn injury, and the effects of estradiol on these levels. Using an experimental animal model, we studied the acute effects of a full-thickness third degree burn on brain levels of TNF-alpha, IL-1beta, and IL-6 and the protective effects of acute estrogen treatment on these levels. Additionally, the acute administration of estrogen on regulation of inflammatory and apoptotic events in the brain following severe burn injury were studied through measuring the levels of phospho-ERK, phospho-Akt, active caspase-3, and PARP cleavage in the placebo and estrogen treated groups. METHODS: In this study, 149 adult Sprague-Dawley male rats received 3rd degree 40% total body surface area (TBSA) burns. Fifteen minutes following burn injury, the animals received a subcutaneous injection of either placebo (n = 72) or 17 beta-estradiol (n = 72). Brains were harvested at 0.5, 1, 2, 4, 6, 8, 12, 18, and 24 hours after injury from the control (n = 5), placebo (n = 8/time point), and estrogen treated animals (n = 8/time point). The brain cytokine levels were measured using the ELISA method. In addition, we assessed the levels of phosphorylated-ERK, phosphorylated-Akt, active caspase-3, and the levels of cleaved PARP at the 24 hour time-point using Western blot analysis. RESULTS: In burned rats, 17 beta-estradiol significantly decreased the levels of brain tissue TNF-alpha (approximately 25%), IL-1beta (approximately 60%), and IL-6 (approximately 90%) when compared to the placebo group. In addition, we determined that in the estrogen-treated rats there was an increase in the levels of phospho-ERK (p < 0.01) and Akt (p < 0.05) at the 24 hour time-point, and that 17 beta-estradiol blocked the activation of caspase-3 (p < 0.01) and subsequent cleavage of PARP (p < 0.05). CONCLUSION: Following severe burn injury, estrogens decrease both brain inflammation and the activation of apoptosis, represented by an increase in the levels of phospho-Akt and inhibition of caspase-3 activation and PARP cleavage. Results from these studies will help further our understanding of how estrogens protect the brain following burn injury, and may provide a novel, safe, and effective clinical treatment to combat remote secondary burn injury in the brain and to preserve cognition.

Activation of a membrane-associated androgen receptor promotes cell death in primary cortical astrocytes.

In the central nervous system, androgens can exert either protective or damage-promoting effects. For example, testosterone protects neurons against beta-amyloid toxicity, whereas in other studies, testosterone exacerbated stroke-induced lesion size. The mechanism underlying this duality of androgens is still unclear. Recently, our laboratory reported that androgens elicit opposite effects on the ERK/MAPK and Akt signaling pathways, depending on whether a membrane androgen receptor (AR) or intracellular AR was activated. By extension, we hypothesized that androgens may affect cell viability differently depending on which receptor is activated. Here, we found that dihydrotestosterone (DHT) protected primary cortical astrocytes from the metabolic and oxidative insult associated with iodoacetic acid-induced toxicity, whereas DHT-BSA, a cell impermeable analog of DHT that preferentially targets the membrane AR, suppressed Akt signaling, increased caspase 3/7 activity, and enhanced iodoacetic acid-induced cell death. Interestingly, DHT-BSA also blocked the protective effects of DHT and estradiol. Collectively, these data support the existence of two, potentially competing, pathways for androgens in a given cell or tissue that may provide insight into the controversy of whether androgen therapy is beneficial or detrimental.

Bacillus tequilensis sp. nov., isolated from a 2000-year-old Mexican shaft-tomb, is closely related to Bacillus subtilis.

A Gram-positive, spore-forming bacillus was isolated from a sample taken from an approximately 2000-year-old shaft-tomb located in the Mexican state of Jalisco, near the city of Tequila. Tentative identification using conventional biochemical analysis consistently identified the isolate as Bacillus subtilis. DNA isolated from the tomb isolate, strain 10b(T), and closely related species was used to amplify a Bacillus-specific portion of the highly conserved 16S rRNA gene and an internal region of the superoxide dismutase gene (sodA(int)). Trees derived from maximum-likelihood methods applied to the sodA(int) sequences yielded non-zero branch lengths between strain 10b(T) and its closest relative, whereas a comparison of a Bacillus-specific 546 bp amplicon of the 16S rRNA gene demonstrated 99 % similarity with B. subtilis. Although the 16S rRNA gene sequences of strain 10b(T) and B. subtilis were 99 % similar, PFGE of NotI-digested DNA of strain 10b(T) revealed a restriction profile that was considerably different from those of B. subtilis and other closely related species. Whereas qualitative differences in whole-cell fatty acids were not observed, significant quantitative differences were found to exist between strain 10b(T) and each of the other closely related Bacillus species examined. In addition, DNA-DNA hybridization studies demonstrated that strain 10b(T) had a relatedness value of less than 70 % with B. subtilis and other closely related species. Evidence from the sodA(int) sequences, whole-cell fatty acid profiles and PFGE analysis, together with results from DNA-DNA hybridization studies, justify the classification of strain 10b(T) as representing a distinct species, for which the name Bacillus tequilensis sp. nov. is proposed. The type strain is 10b(T) (=ATCC BAA-819(T)=NCTC 13306(T)).

Dihydrotestosterone differentially modulates the mitogen-activated protein kinase and the phosphoinositide 3-kinase/Akt pathways through the nuclear and novel membrane androgen receptor in C6 cells.

Androgens such as dihydrotestosterone (DHT) are known to exert their effects through the activation of intracellular receptors that regulate the transcription of target genes. Alternatively, nongenomic mechanisms, including the activation of such signaling pathways as the MAPK pathways, have been described. It is unclear, however, whether this latter mechanism of action is mediated by the classical androgen receptor (AR) or some alternative mechanism. In this study, using a glial cell model (C6 cells) that we found to express the AR, we identified that DHT increased the phosphorylation of both ERK and Akt, key effectors of the neuroprotection-associated MAPK and phosphoinositide 3-kinase signaling pathways, respectively, and ERK phosphorylation was blocked by the AR antagonist, flutamide. In contrast, the membrane-impermeable, BSA-conjugated androgen (DHT-BSA) caused a dose-dependent suppression of ERK and Akt phosphorylation, suggesting the existence of a novel membrane-associated AR that mediates this opposite effect on neuroprotective signaling. This is also supported by the observation of DHT-displaceable binding sites on the cell surface of live C6 cells. Collectively, these data support the existence of a novel membrane-associated AR in glial cells and argue for the existence of two, potentially competing, pathways in a given cell or tissue. This mutual antagonism was supported by the ability of DHT-BSA to attenuate DHT-induced ERK phosphorylation. Thus, depending on the predominance of one receptor mechanism over another, the outcome of androgen treatment may be very different and, as such, could help explain existing discrepancies as to whether androgens are protective or damage inducing.