Pharmacological Characterization of a Novel Neuroactive Steroid Prodrug, NTS-104, and Its Neuroprotective Activity in Experimental Stroke Models.

BACKGROUND: Ischemic stroke affects about 700 000 patients per year in the United States, and to date, there are no effective pharmacological agents that promote recovery. Here, we studied the pharmacokinetics, pharmacodynamics, and efficacy of NTS-105, a novel neuroactive steroid, and NTS-104, a prodrug of NTS-105, in 2 models of ischemic stroke. METHODS: The pharmacodynamics and pharmacokinetics of NTS-104/105 were investigated in naive and stroke rats, and models of embolic and transient middle cerebral artery occlusion were used to investigate the dose-related effects of NTS-104. All rats were randomly assigned into the experimental groups, and all outcome measurements were performed blindly. RESULTS: Blood plasma and brain pharmacokinetic analysis revealed that NTS-104 rapidly converted to NTS-105, which reached peak concentration at ≈1 hour after dosing and distributed similarly to normal and ischemic brains. NTS-104 administration 4 hours after embolic middle cerebral artery occlusion led to a dose-dependent improvement of neurological outcomes and a dose-dependent reduction of infarct volumes relative to vehicle-treated animals. A single dose level study confirmed that NTS-104 administered 4 hours after transient middle cerebral artery occlusion was also neuroprotective. Quantitative ELISA revealed that NTS-104 treatment resulted in time- and dose-dependent changes in AKT activation and cytokine levels within the ischemic brain, which included reductions of IL-6, VEGF, ICAM-1, IL-1ß, MCP-1, RAGE, and GM-CSF. Time- and dose-dependent reductions in IL-6 and GM-CSF were also observed in the plasma along with an elevation of galectin-1. CONCLUSIONS: NTS-104 is a novel prodrug that converts to a novel neuroactive steroid, NTS-105, which improves functional outcomes in experimental ischemic stroke models.

Sensitivity of unilateral- versus bilateral-onset spike-wave discharges to ethosuximide and carbamazepine in the fluid percussion injury rat model of traumatic brain injury.

Unilateral-onset spike-wave discharges (SWDs) following fluid percussion injury (FPI) in rats have been used for nearly two decades as a model for complex partial seizures in human posttraumatic epilepsy (PTE). This study determined if SWDs with a unilateral versus bilateral cortical onset differed. In this experiment, 2-mo-old rats received severe FPI (3 atm) or sham surgery and were instrumented for chronic video-electrocorticography (ECoG) recording (up to 9 mo). The antiseizure drug, carbamazepine (CBZ), and the antiabsence drug, ethosuximide (ETX), were administered separately to determine if they selectively suppressed unilateral- versus bilateral-onset SWDs, respectively. SWDs did not significantly differ between FPI and sham rats on any measured parameter (wave-shape, frequency spectrum, duration, or age-related progression), including unilateral (∼17%) versus bilateral (∼83%) onsets. SWDs with a unilateral onset preferentially originated ipsilateral to the craniotomy in both FPI and sham rats, suggesting that the unilateral-onset SWDs were related to surgical injury and not specifically to FPI. ETX profoundly suppressed SWDs with either unilateral or bilateral onsets, and CBZ had no effect on either type of SWD. These results suggest that SWDs with either a unilateral or bilateral onset have a pharmacosensitivity similar to absence seizures and are very different from the complex partial seizures of PTE. Therefore, SWDs with a unilateral onset after FPI are not a model of the complex partial seizures that occur in PTE, and their use for finding new treatments for PTE could be counterproductive, particularly if their close similarity to normal brain oscillations is not acknowledged.NEW & NOTEWORTHY Unilateral-onset spike-wave discharges (SWDs) in rats have been used to model complex partial seizures in human posttraumatic epilepsy (PTE), compared to bilateral-onset SWDs thought to reflect human absence seizures. Here, we show that both unilateral- and bilateral-onset SWDs following traumatic brain injury are suppressed by the antiabsence drug ethosuximide and are unaffected by the antiseizure drug carbamazepine. We propose that unilateral-onset SWDs are not useful for studying mechanisms of, or treatments for, PTE.

IonStar enables high-precision, low-missing-data proteomics quantification in large biological cohorts.

Reproducible quantification of large biological cohorts is critical for clinical/pharmaceutical proteomics yet remains challenging because most prevalent methods suffer from drastically declined commonly quantified proteins and substantially deteriorated quantitative quality as cohort size expands. MS2-based data-independent acquisition approaches represent tremendous advancements in reproducible protein measurement, but often with limited depth. We developed IonStar, an MS1-based quantitative approach enabling in-depth, high-quality quantification of large cohorts by combining efficient/reproducible experimental procedures with unique data-processing components, such as efficient 3D chromatographic alignment, sensitive and selective direct ion current extraction, and stringent postfeature generation quality control. Compared with several popular label-free methods, IonStar exhibited far lower missing data (0.1%), superior quantitative accuracy/precision [∼5% intragroup coefficient of variation (CV)], the widest protein abundance range, and the highest sensitivity/specificity for identifying protein changes (<5% false altered-protein discovery) in a benchmark sample set (n = 20). We demonstrated the usage of IonStar by a large-scale investigation of traumatic injuries and pharmacological treatments in rat brains (n = 100), quantifying >7,000 unique protein groups (>99.8% without missing data across the 100 samples) with a low false discovery rate (FDR), two or more unique peptides per protein, and high quantitative precision. IonStar represents a reliable and robust solution for precise and reproducible protein measurement in large cohorts.

Potential Neuroprotective Mechanisms of Methamphetamine Treatment in Traumatic Brain Injury Defined by Large-Scale IonStar-Based Quantitative Proteomics.

Although traumatic brain injury (TBI) causes hospitalizations and mortality worldwide, there are no approved neuroprotective treatments, partly due to a poor understanding of the molecular mechanisms underlying TBI neuropathology and neuroprotection. We previously reported that the administration of low-dose methamphetamine (MA) induced significant functional/cognitive improvements following severe TBI in rats. We further demonstrated that MA mediates neuroprotection in part, via dopamine-dependent activation of the PI3K-AKT pathway. Here, we further investigated the proteomic changes within the rat cortex and hippocampus following mild TBI (TM), severe TBI (TS), or severe TBI plus MA treatment (TSm) compared to sham operated controls. We identified 402 and 801 altered proteins (APs) with high confidence in cortical and hippocampal tissues, respectively. The overall profile of APs observed in TSm rats more closely resembled those seen in TM rather than TS rats. Pathway analysis suggested beneficial roles for acute signaling through IL-6, TGFß, and IL-1ß. Moreover, changes in fibrinogen levels observed in TSm rats suggested a potential role for these proteins in reducing/preventing TBI-induced coagulopathies. These data facilitate further investigations to identify specific pathways and proteins that may serve as key targets for the development of neuroprotective therapies.

Visualization of thalamic calcium influx with quantitative susceptibility mapping as a potential imaging biomarker for repeated mild traumatic brain injury.

A key event in the pathophysiology of traumatic brain injury (TBI) is the influx of substantial amounts of Ca2+ into neurons, particularly in the thalamus. Detection of this calcium influx in vivo would provide a window into the biochemical mechanisms of TBI with potentially significant clinical implications. In the present work, our central hypothesis was that the Ca2+ influx could be imaged in vivo with the relatively recent MRI technique of quantitative susceptibility mapping (QSM). Wistar rats were divided into five groups: naive controls, sham-operated experimental controls, single mild TBI, repeated mild TBI, and single severe TBI. We employed the lateral fluid percussion injury (FPI) model, which replicates clinical TBI without skull fracture, performed 9.4 Tesla MRI with a 3D multi-echo gradient-echo sequence at weeks 1 and 4 post-injury, computed susceptibility maps using V-SHARP and the QUASAR-HEIDI technique, and performed histology. Sham, experimental controls animals, and injured animals did not demonstrate calcifications at 1 week after the injury. At week 4, calcifications were found in the ipsilateral thalamus of 25-50% of animals after a single TBI and 83% of animals after repeated mild TBI. The location and appearance of calcifications on stained sections was consistent with the appearance on the in vivo susceptibility maps (correlation of volumes: r = 0.7). Our findings suggest that persistent calcium deposits represent a primary pathology of repeated injury and that FPI-QSM has the potential to become a sensitive tool for studying pathophysiology related to mild TBI in vivo.

Extracellular Vesicles as Diagnostics and Therapeutics for Structural Epilepsies.

Extracellular vesicles (EVs) are small vesicles involved in intercellular communication. Data is emerging that EVs and their cargo have potential as diagnostic biomarkers and treatments for brain diseases, including traumatic brain injury and epilepsy. Here, we summarize the current knowledge regarding changes in EV numbers and cargo in status epilepticus (SE) and traumatic brain injury (TBI), which are clinically significant etiologies for acquired epileptogenesis in animals and humans. We also review encouraging data, which suggests that EVs secreted by stem cells may serve as recovery-enhancing treatments for SE and TBI. Using Gene Set Enrichment Analysis, we show that brain EV-related transcripts are positively enriched in rodent models of epileptogenesis and epilepsy, and altered in response to anti-seizure drugs. These data suggest that EVs show promise as biomarkers, treatments and drug targets for epilepsy. In parallel to gathering conceptual knowledge, analytics platforms for the isolation and analysis of EV contents need to be further developed.

In Vitro and In Vivo Pipeline for Validation of Disease-Modifying Effects of Systems Biology-Derived Network Treatments for Traumatic Brain Injury-Lessons Learned.

We developed a pipeline for the discovery of transcriptomics-derived disease-modifying therapies and used it to validate treatments in vitro and in vivo that could be repurposed for TBI treatment. Desmethylclomipramine, ionomycin, sirolimus and trimipramine, identified by in silico LINCS analysis as candidate treatments modulating the TBI-induced transcriptomics networks, were tested in neuron-BV2 microglial co-cultures, using tumour necrosis factor α as a monitoring biomarker for neuroinflammation, nitrite for nitric oxide-mediated neurotoxicity and microtubule associated protein 2-based immunostaining for neuronal survival. Based on (a) therapeutic time window in silico, (b) blood-brain barrier penetration and water solubility, (c) anti-inflammatory and neuroprotective effects in vitro (p < 0.05) and (d) target engagement of Nrf2 target genes (p < 0.05), desmethylclomipramine was validated in a lateral fluid-percussion model of TBI in rats. Despite the favourable in silico and in vitro outcomes, in vivo assessment of clomipramine, which metabolizes to desmethylclomipramine, failed to demonstrate favourable effects on motor and memory tests. In fact, clomipramine treatment worsened the composite neuroscore (p < 0.05). Weight loss (p < 0.05) and prolonged upregulation of plasma cytokines (p < 0.05) may have contributed to the worsened somatomotor outcome. Our pipeline provides a rational stepwise procedure for evaluating favourable and unfavourable effects of systems-biology discovered compounds that modulate post-TBI transcriptomics.

Intracranial pressure changes after mild traumatic brain injury: a systematic review.

OBJECTIVE: Intracranial pressure (ICP) after mild traumatic brain injury (mTBI) is poorly studied due to lack of sensitive non-invasive methods. The purpose of this review was to summarize the existing knowledge of changes in ICP after mTBI. Literature selection: PubMed, Embase, CINAHL, and Scopus were searched by three reviewers independently up to December 2016. INCLUSION CRITERIA: animal and human studies measuring ICP and brain oedema after an mTBI. EXCLUSION CRITERIA: moderate and severe forms of traumatic brain injury, repeat samples, and studies that measured ICP at the time of impact but not after. Study quality was assessed using Downs and Black criteria. RESULTS: Of 1067 papers, 9 studies were included. In human studies, one provided direct evidence on increased, one provided indirect evidence of increased, and two provided indirect evidence of decreased ICP. In animal studies, three studies provided direct evidence of increased, one provided indirect evidence of increased, and one provided indirect evidence of no change in ICP. CONCLUSION: The existing research suggests that there may be increased ICP after mTBI and animal studies suggest an elevation for days which returns to baseline, which corresponds with functional and symptomatic recovery. Future human studies using sensitive indirect methods to measure ICP longitudinally after mTBI are needed.

GLT1 overexpression reverses established neuropathic pain-related behavior and attenuates chronic dorsal horn neuron activation following cervical spinal cord injury.

Development of neuropathic pain occurs in a major portion of traumatic spinal cord injury (SCI) patients, resulting in debilitating and often long-term physical and psychological burdens. Following SCI, chronic dysregulation of extracellular glutamate homeostasis has been shown to play a key role in persistent central hyperexcitability of superficial dorsal horn neurons that mediate pain neurotransmission, leading to various forms of neuropathic pain. Astrocytes express the major CNS glutamate transporter, GLT1, which is responsible for the vast majority of functional glutamate uptake, particularly in the spinal cord. In our unilateral cervical contusion model of mouse SCI that is associated with ipsilateral forepaw heat hypersensitivity (a form of chronic at-level neuropathic pain-related behavior), we previously reported significant and long-lasting reductions in GLT1 expression and functional GLT1-mediated glutamate uptake in cervical spinal cord dorsal horn. To therapeutically address GLT1 dysfunction following cervical contusion SCI, we injected an adeno-associated virus type 8 (AAV8)-Gfa2 vector into the superficial dorsal horn to increase GLT1 expression selectively in astrocytes. Compared to both contusion-only animals and injured mice that received AAV8-eGFP control injection, AAV8-GLT1 delivery increased GLT1 protein expression in astrocytes of the injured cervical spinal cord dorsal horn, resulting in a significant and persistent reversal of already-established heat hypersensitivity. Furthermore, AAV8-GLT1 injection significantly reduced expression of the transcription factor and marker of persistently increased neuronal activation, ΔFosB, in superficial dorsal horn neurons. These results demonstrate that focal restoration of GLT1 expression in the superficial dorsal horn is a promising target for treating chronic neuropathic pain following SCI.

Transplantation of glial progenitors that overexpress glutamate transporter GLT1 preserves diaphragm function following cervical SCI.

Approximately half of traumatic spinal cord injury (SCI) cases affect cervical regions, resulting in chronic respiratory compromise. The majority of these injuries affect midcervical levels, the location of phrenic motor neurons (PMNs) that innervate the diaphragm. A valuable opportunity exists following SCI for preventing PMN loss that occurs during secondary degeneration. One of the primary causes of secondary injury is excitotoxicity due to dysregulation of extracellular glutamate homeostasis. Astrocytes express glutamate transporter 1 (GLT1), which is responsible for the majority of CNS glutamate clearance. Given our observations of GLT1 dysfunction post-SCI, we evaluated intraspinal transplantation of Glial-Restricted Precursors (GRPs)--a class of lineage-restricted astrocyte progenitors--into ventral horn following cervical hemicontusion as a novel strategy for reconstituting GLT1 function, preventing excitotoxicity and protecting PMNs in the acutely injured spinal cord. We find that unmodified transplants express low levels of GLT1 in the injured spinal cord. To enhance their therapeutic properties, we engineered GRPs with AAV8 to overexpress GLT1 only in astrocytes using the GFA2 promoter, resulting in significantly increased GLT1 protein expression and functional glutamate uptake following astrocyte differentiation in vitro and after transplantation into C4 hemicontusion. Compared to medium-only control and unmodified GRPs, GLT1-overexpressing transplants reduced lesion size, diaphragm denervation and diaphragm dysfunction. Our findings demonstrate transplantation-based replacement of astrocyte GLT1 is a promising approach for SCI.

Hilar mossy cells provide the first glutamatergic synapses to adult-born dentate granule cells.

Adult-generated granule cells (GCs) in the dentate gyrus must establish synapses with preexisting neurons to participate in network activity. To determine the source of early glutamatergic synapses on newborn GCs in adult mice, we examined synaptic currents at the developmental stage when NMDA receptor-mediated silent synapses are first established. We show that hilar mossy cells provide initial glutamatergic synapses as well as disynaptic GABAergic input to adult-generated dentate GCs.

Overexpression of the astrocyte glutamate transporter GLT1 exacerbates phrenic motor neuron degeneration, diaphragm compromise, and forelimb motor dysfunction following cervical contusion spinal cord injury.

A major portion of spinal cord injury (SCI) cases affect midcervical levels, the location of the phrenic motor neuron (PhMN) pool that innervates the diaphragm. While initial trauma is uncontrollable, a valuable opportunity exists in the hours to days following SCI for preventing PhMN loss and consequent respiratory dysfunction that occurs during secondary degeneration. One of the primary causes of secondary injury is excitotoxic cell death due to dysregulation of extracellular glutamate homeostasis. GLT1, mainly expressed by astrocytes, is responsible for the vast majority of functional uptake of extracellular glutamate in the CNS, particularly in spinal cord. We found that, in bacterial artificial chromosome-GLT1-enhanced green fluorescent protein reporter mice following unilateral midcervical (C4) contusion SCI, numbers of GLT1-expressing astrocytes in ventral horn and total intraspinal GLT1 protein expression were reduced soon after injury and the decrease persisted for ≥6 weeks. We used intraspinal delivery of adeno-associated virus type 8 (AAV8)-Gfa2 vector to rat cervical spinal cord ventral horn for targeting focal astrocyte GLT1 overexpression in areas of PhMN loss. Intraspinal delivery of AAV8-Gfa2-GLT1 resulted in transduction primarily of GFAP(+) astrocytes that persisted for ≥6 weeks postinjury, as well as increased intraspinal GLT1 protein expression. Surprisingly, we found that astrocyte-targeted GLT1 overexpression increased lesion size, PhMN loss, phrenic nerve axonal degeneration, and diaphragm neuromuscular junction denervation, and resulted in reduced functional diaphragm innervation as assessed by phrenic nerve-diaphragm compound muscle action potential recordings. These results demonstrate that GLT1 overexpression via intraspinal AAV-Gfa2-GLT1 delivery exacerbates neuronal damage and increases respiratory impairment following cervical SCI.

GLT1 overexpression in SOD1(G93A) mouse cervical spinal cord does not preserve diaphragm function or extend disease.

Amyotrophic lateral sclerosis (ALS) is characterized by relatively rapid degeneration of both upper and lower motor neurons, with death normally occurring 2-5years following diagnosis primarily due to respiratory paralysis resulting from phrenic motor neuron (PhMN) loss and consequent diaphragm denervation. In ALS, cellular abnormalities are not limited to MNs. For example, decreased levels and aberrant functioning of the major central nervous system (CNS) glutamate transporter, GLT1, occur in spinal cord and motor cortex astrocytes of both humans with ALS and in SOD1(G93A) rodents, a widely studied ALS animal model. This results in dysregulation of extracellular glutamate homeostasis and consequent glutamate excitotoxicity, a primary mechanism responsible for MN loss in ALS animal models and in the human disease. Given these observations of GLT1 dysfunction in areas of MN loss, as well as the importance of testing therapeutic strategies for preserving PhMNs in ALS, we evaluated intraspinal delivery of an adeno-associated virus type 8 (AAV8)-Gfa2 vector to the cervical spinal cord ventral horn of SOD1(G93A) ALS mice for focally restoring intraspinal GLT1 expression. AAV8 was specifically injected into the ventral horn bilaterally throughout the cervical enlargement at 110days of age, a clinically-relevant time point coinciding with phenotypic/symptomatic disease onset. Intraspinal delivery of AAV8-Gfa2-GLT1 resulted in robust transduction primarily of GFAP(+) astrocytes that persisted until disease endstage, as well as a 2-3-fold increase in total intraspinal GLT1 protein expression in the ventral horn. Despite this robust level of astrocyte transduction and GLT1 elevation, GLT1 overexpression did not protect PhMNs, preserve histological PhMN innervation of the diaphragm NMJ, or prevent decline in diaphragmatic respiratory function as assessed by phrenic nerve-diaphragm compound muscle action potential (CMAP) recordings compared to control AAV8-Gfa2-eGFP injected mice. In addition, AAV-Gfa2-GLT1 did not delay forelimb disease onset, extend disease duration (i.e. time from either forelimb or hindlimb disease onsets to endstage) or prolong overall animal survival. These findings suggest that focal restoration of GLT1 expression in astrocytes of the cervical spinal cord using AAV delivery is not an effective therapy for ALS.

Recent advances in the treatment of corneal ectasia with intrastromal corneal ring segments.

PURPOSE OF REVIEW: To review the recent advances and reported outcomes in the use of intrastromal corneal ring segments (ICRS) for the treatment of corneal ectasia. RECENT FINDINGS: ICRS are a well-tolerated and effective treatment for patients with corneal ectasia, particularly keratoconus, offering long-term improvement in visual, refractive, and keratometric measures. ICRS do not consistently decrease corneal aberrations. Patients with mild-to-moderate keratoconus, known to have less predictable outcomes with ICRS, may be better selected and treated with the use of customized nomograms, accounting for factors such as internal astigmatism. Corneal collagen cross-linking performed after ICRS implantation is an important complementary treatment in preventing the progression of ectasia, whereas subsequent treatment with either photorefractive keratectomy or toric intraocular lens implantation offers a significantly improved visual and refractive result. SUMMARY: ICRS are an important component to the treatment of corneal ectasia. Knowledge of outcomes among specific groups of patients should improve treatment planning and nomograms. Combined treatments with ICRS allow for notable improvements in corneal stability and refractive error, in addition to the improvement in irregular astigmatism seen with ICRS.

Phenoxybenzamine is neuroprotective in a rat model of severe traumatic brain injury.

Phenoxybenzamine (PBZ) is an FDA approved α-1 adrenergic receptor antagonist that is currently used to treat symptoms of pheochromocytoma. However, it has not been studied as a neuroprotective agent for traumatic brain injury (TBI). While screening neuroprotective candidates, we found that phenoxybenzamine reduced neuronal death in rat hippocampal slice cultures following exposure to oxygen glucose deprivation (OGD). Using this system, we found that phenoxybenzamine reduced neuronal death over a broad dose range (0.1 µM-1 mM) and provided efficacy when delivered up to 16 h post-OGD. We further tested phenoxybenzamine in the rat lateral fluid percussion model of TBI. When administered 8 h after TBI, phenoxybenzamine improved neurological severity scoring and foot fault assessments. At 25 days post injury, phenoxybenzamine treated TBI animals also showed a significant improvement in both learning and memory compared to saline treated controls. We further examined gene expression changes within the cortex following TBI. At 32 h post-TBI phenoxybenzamine treated animals had significantly lower expression of pro-inflammatory signaling proteins CCL2, IL1ß, and MyD88, suggesting that phenoxybenzamine may exert a neuroprotective effect by reducing neuroinflammation after TBI. These data suggest that phenonxybenzamine may have application in the treatment of TBI.

Ursodiol and colorectal cancer or dysplasia risk in primary sclerosing cholangitis and inflammatory bowel disease: a meta-analysis.

BACKGROUND: Patients with primary sclerosing cholangitis (PSC) and colonic inflammatory bowel disease (IBD) demonstrate increased risk of colorectal cancer. Prior studies have yielded conflicting information on the relationship between ursodiol (UDCA) and the risk of colorectal cancer or dysplasia in this group. AIMS: To examine the impact of UDCA on risk of colorectal cancer or dysplasia in adult PSC and IBD patients. METHODS: A systematic review and meta-analysis of case-control and cohort studies was performed. Subgroup analysis compared the effects of "low-to-medium" (<25 mg/kg/day) versus "high" dose (≥ 25 mg/kg/day) UDCA exposures. RESULTS: Inclusion and exclusion criteria, as well as all variables, were determined a priori. Seven papers, with 707 participants and greater than 5,751 person-years of follow-up time, met the criteria for final analysis. The overall pooled relative risk using a random effects model was not statistically significant (RR = 0.87, 95 % CI 0.51-1.49, p = 0.62). Subgroup analysis by UDCA dose category in a random effects model was not statistically significant (RR = 0.64, 95 % CI 0.38-1.07, p = 0.09), but suggested a possible trend in risk reduction at low-to-medium-dose exposures that may warrant further investigation. CONCLUSION: UDCA use was not associated with risk of colorectal cancer or dysplasia in adult PSC and IBD patients, but UDCA dose was a source of heterogeneity across studies. Subgroup analysis suggests a possible trend toward decreased colorectal cancer risk in low-to-medium-dose exposures. Additional study of UDCA treatments at low doses in PSC and IBD patients may be warranted.

High-quality and robust protein quantification in large clinical/pharmaceutical cohorts with IonStar proteomics investigation.

Robust, reliable quantification of large sample cohorts is often essential for meaningful clinical or pharmaceutical proteomics investigations, but it is technically challenging. When analyzing very large numbers of samples, isotope labeling approaches may suffer from substantial batch effects, and even with label-free methods, it becomes evident that low-abundance proteins are not reliably measured owing to unsufficient reproducibility for quantification. The MS1-based quantitative proteomics pipeline IonStar was designed to address these challenges. IonStar is a label-free approach that takes advantage of the high sensitivity/selectivity attainable by ultrahigh-resolution (UHR)-MS1 acquisition (e.g., 120-240k full width at half maximum at m/z = 200) which is now widely available on ultrahigh-field Orbitrap instruments. By selectively and accurately procuring quantitative features of peptides within precisely defined, very narrow m/z windows corresponding to the UHR-MS1 resolution, the method minimizes co-eluted interferences and substantially enhances signal-to-noise ratio of low-abundance species by decreasing noise level. This feature results in high sensitivity, selectivity, accuracy and precision for quantification of low-abundance proteins, as well as fewer missing data and fewer false positives. This protocol also emphasizes the importance of well-controlled, robust experimental procedures to achieve high-quality quantification across a large cohort. It includes a surfactant cocktail-aided sample preparation procedure that achieves high/reproducible protein/peptide recoveries among many samples, and a trapping nano-liquid chromatography-mass spectrometry strategy for sensitive and reproducible acquisition of UHR-MS1 peptide signal robustly across a large cohort. Data processing and quality evaluation are illustrated using an example dataset ( http://proteomecentral.proteomexchange.org ), and example results from pharmaceutical project and one clinical project (patients with acute respiratory distress syndrome) are shown. The complete IonStar pipeline takes ~1-2 weeks for a sample cohort containing ~50-100 samples.

Differential Patterns of Change in Brain Connectivity Resulting from Severe Traumatic Brain Injury.

Background: Traumatic brain injury (TBI) damages white matter tracts, disrupting brain network structure and communication. There exists a wide heterogeneity in the pattern of structural damage associated with injury, as well as a large heterogeneity in behavioral outcomes. However, little is known about the relationship between changes in network connectivity and clinical outcomes. Materials and Methods: We utilize the rat lateral fluid-percussion injury model of severe TBI to study differences in brain connectivity in 8 animals that received the insult and 11 animals that received only a craniectomy. Diffusion tensor imaging is performed 5 weeks after the injury and network theory is used to investigate changes in white matter connectivity. Results: We find that (1) global network measures are not able to distinguish between healthy and injured animals; (2) injury induced alterations predominantly exist in a subset of connections (subnetworks) distributed throughout the brain; and (3) injured animals can be divided into subgroups based on changes in network motifs-measures of local structural connectivity. In addition, alterations in predicted functional connectivity indicate that the subgroups have different propensities to synchronize brain activity, which could relate to the heterogeneity of clinical outcomes. Discussion: These results suggest that network measures can be used to quantify progressive changes in brain connectivity due to injury and differentiate among subpopulations with similar injuries, but different pathological trajectories.

Characteristics of ocular trauma in the United States.

PURPOSE: We aimed to study the characteristics of ocular trauma, an important but largely preventable global cause of blindness, in the United States. METHODS: Retrospective chart review of the National Trauma Data Bank (2008-2014) was performed. All patients with ocular trauma were identified using ICD-9CM codes. The collected data were statistically analyzed with student's t-test, Chi-squared test, and logistic regression analysis performed using the SPSS software. The significance was set at p<0.05. RESULTS: It was found that 316,485 (5.93%) of the 5,336,575 admitted trauma patients had ocular injuries. Their mean (SD) age was 41.8 (23) years, and most of them were men (69.4%). Race/ethnicity distribution was White 66.1%, Black 15.1%, and Hispanic 12.3%. The common injuries were orbital 39.5% and eye/adnexa contusions 34%. Associated traumatic brain injury was present in 58.2%. The frequent mechanisms were falls 25.5%, motor vehicle accident-occupant 21.8%, and struck by/against 17.6%. Patients <21 years of age had higher odds of cut/pierce injuries (OR=3.29, 95%CI=3.07-3.51) than the other age groups, those aged 21-64 years had higher odds of motor vehicle accident-cyclist (OR=4.95, 95%CI=4.71-5.19), and those >65 years had higher odds of falls (OR=16.75, 95%CI=16.39-17.12); p<0.001. The Blacks had a greater likelihood of firearm injuries (OR=3.24, 95%CI=3.10-3.39) than the other racial/ethnic groups, the Hispanics experienced more of cut/pierce injuries (OR=2.01, 95%CI=1.85-2.18), and the Whites experienced more of falls (OR=2.3, 95%CI=2.3-2.4); p<0.001. The Blacks (OR=3.41, 95%CI=3.34-3.48) and Hispanics (OR=1.75, 95%CI=1.71-1.79) mostly suffered assaults, while the Whites suffered unintentional injuries (OR=2.78 95%CI=2.74-2.84); p<0.001. Optic nerve/visual pathway injuries had the greatest association with very severe injury severity scores (OR=3.27, 95%CI=3.05-3.49) and severe Glasgow Coma Scores (OR=3.30, 95%CI=3.08-3.54); p<0.001. The mortality rate was 3.9%. CONCLUSIONS: Male preponderance and falls, motor vehicle accident-occupant, and struck by/against mechanisms agree with the previous reports. The identified demographic patterns underscore the need to develop group-specific preventive measures.

Increased p38 mitogen-activated protein kinase signaling is involved in the oxidative stress associated with oxygen and glucose deprivation in neonatal hippocampal slice cultures.

The pathological basis of neonatal hypoxia-ischemia (HI) brain damage is characterized by neuronal cell loss. Oxidative stress is thought to be one of the main causes of HI-induced neuronal cell death. The p38 mitogen-activated protein kinase (MAPK) is activated under conditions of cell stress. However, its pathogenic role in regulating the oxidative stress associated with HI injury in the brain is not well understood. Thus, this study was conducted to examine the role of p38 MAPK signaling in neonatal HI brain injury using neonatal rat hippocampal slice cultures exposed to oxygen/glucose deprivation (OGD). Our results indicate that OGD led to a transient increase in p38 MAPK activation that preceded increases in superoxide generation and neuronal death. This increase in neuronal cell death correlated with an increase in the activation of caspase-3 and the appearance of apoptotic neuronal cells. Pre-treatment of slice cultures with the p38 MAPK inhibitor, SB203580, or the expression of an antisense p38 MAPK construct only in neuronal cells, through a Synapsin I-1-driven adeno-associated virus vector, inhibited p38 MAPK activity and exerted a neuroprotective effect as demonstrated by decreases in OGD-mediated oxidative stress, caspase activation and neuronal cell death. Thus, we conclude that the activation of p38 MAPK in neuronal cells plays a key role in the oxidative stress and neuronal cell death associated with OGD.