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.

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.

Characteristics of ocular trauma in the United States / Características de trauma ocular nos Estados Unidos

ABSTRACT 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.

RESUMO Objetivo: O trauma ocular é uma causa importante e amplamente evitável de cegueira em todo o mundo. Nosso objetivo é estudar suas características nos EUA. Métodos: Revisão retrospectiva do National Trauma Data Bank (2008-2014). Todos os pacientes com trauma ocular foram identificados com códigos CID-9CM. Os dados coletados foram analisados estatísticamente e o teste t de student foi utilizado. As análises qui-quadrado e de regressão logística foram realizadas com o software SPSS. A significância foi estabelecida em p<0,05. Resultados: 316.485 (5,93%) de 5.336.575 pacientes internados com trauma, apresentaram lesões oculares. A média (DP) de idade foi de 41,8 (23) anos. A maioria era do sexo masculino (69,4%). A distribuição raça/etnia foi branca: 66,1%, negra; 15,1% e hispânica: 12,3%. As lesões comuns foram orbitárias: 39,5% e contusões dos olhos/anexos: 34%. A maioria (58,2%) teve lesão cerebral traumática. Os mecanismos frequentes foram: quedas: 25,5%, acidente com veículos motorizados: 21,8% e acidentes atingidos por algo/contra algo: 17,6%. Os pacientes <21 anos apresentaram chance aumentada de lesões com corte/perfuração (RC=3,29; IC95% = 3,07-3,51) do que outras faixas etárias, aqueles entre 21-64 anos responderam por acidente automobilístico-ciclista: (RC=4,95; IC95% = 4,71-5,19) e aqueles >65 anos foram vítimas de quedas (RC=16,75; IC 95% = 16,39-17,12); p<0,001. Os negros apresentaram maior probabilidade de lesões por arma de fogo (RC=3,24; IC95% = 3,10-3,39) do que outras raças/etnias e os hispânicos tiveram mais lesões de corte/perfuração (RC=2,01; IC95%= 1,85-2,18) enquanto os brancos tiveram mais quedas: (RC=2,3; IC95%= 2,3-2,4); p<0,001. Os negros (RC=3,41; IC95% = 3,34-3,48) e os hispânicos (RC=1,75; IC95% = 1,71-1,79) sofreram principalmente agressões e os brancos tiveram lesões não intencionais (RC=2,78; IC95% 2,74-2,84); p<0,001. Lesões do nervo óptico/via visual apresentaram maior associação com escore de gravidade de lesão muito grave (RC=3,27; IC95%= 3,05-3,49) e escores graves de Coma de Glasgow (RC=3,30; IC95%= 3,08-3,54); p<0,001. A taxa de mortalidade foi de 3,9%. Conclusões: Houve preponderância masculina a quedas, a acidentes com veículo motorizado atingidos por algo/contra algo conforme os relatos anteriores. Os padrões demográficos identificados realçam a necessidade de desenvolver medidas de prevenção específicas para os diferentes grupos.

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.

Firearm-associated ocular injuries: analysis of national trauma data / Lesões oculares relacionadas a armas de fogo: análise de dados nacionais de traumas nos EUA

ABSTRACT Purpose: The United States of America has the highest gun ownership rate of all high-income nations, and firearms have been identified as a leading cause of ocular trauma and visual impairment. The purpose of this study was to characterize firearm-associated ocular injury and identify at-risk groups. Methods: Patients admitted with firearm-associated ocular injury were identified from the National Trauma Data Bank (2008-2014) using the International Classification of Diseases, Ninth Revision, Clinical Modification diagnostic codes and E-codes for external causes. Statistical analysis was performed using the SPSS 24 software. Significance was set at p<0.05. Results: Of the 235,254 patients, 8,715 (3.7%) admitted with firearm-associated trauma had ocular injuries. Mean (standard deviation) age was 33.8 (16.9) years. Most were males (85.7%), White (46.6%), and from the South (42.9%). Black patients comprised 35% of cases. Common injuries were orbital fractures (38.6%) and open globe injuries (34.7%). Frequent locations of injury were at home (43.8%) and on the street (21.4%). Black patients had the highest risk of experiencing assault (odds ratio [OR]: 9.0; 95% confidence interval [CI]: 8.02-10.11; p<0.001) and street location of injury (OR: 3.05; 95% CI: 2.74-3.39; p<0.001), while White patients had the highest risk of self-­inflicted injury (OR: 10.53; 95% CI: 9.39-11.81; p<0.001) and home location of injury (OR: 3.64; 95% CI: 3.33-3.98; p<0.001). There was a steadily increasing risk of self-inflicted injuries with age peaking in those >80 years (OR: 12.01; 95% CI: 7.49-19.23; p<0.001). Mean (standard deviation) Glasgow Coma Scale and injury severity scores were 10 (5.5) and 18.6 (13.0), respectively. Most injuries (53.1%) were classified as severe or very severe injury, 64.6% had traumatic brain injury, and mortality occurred in 16% of cases. Conclusion: Most firearm-associated ocular injuries occurred in young, male, White, and Southern patients. Blacks were disproportionally affected. Most firearm-associated ocular injuries were sight-­threatening and associated with traumatic brain injury. The majority survived, with potential long-term disabilities. The demographic differences identified in this study may represent potential targets for prevention.

RESUMO Objetivo: Os Estados Unidos têm a maior taxa de posse de armas de fogo de todos os países de alta renda e essas armas foram identificados como uma das maiores causas de trauma ocular e deficiência visual. O objetivo deste estudo foi caracterizar as lesões oculares associadas a armas de fogo e identificar grupos de risco. Métodos: Foram identificados pacientes hospitalizados com lesões oculares associadas a armas de fogo no período de 2008 a 2014, a partir do Banco de Dados Nacional de Trauma (National Trauma Data Bank), usando os códigos de diagnósticos da CID9MC e códigos "E" para causas externas. A análise estatística foi efetuada usando o programa SPSS. O nível de significância considerado foi de p<0,05. Resultados: De um total de 235.254 pacientes hospitalizados com trauma associado a armas de fogo, 8.715 (3,7%) tinham lesões oculares. A média de idade foi de 33,8 (DP 16,9) anos. A maioria foi de homens (85,7%), brancos (46,6%) e da região Sul (42,9%); 35% dos pacientes eram negros. As lesões mais comuns foram fraturas de órbita (38,6%) e lesões de globo aberto (34,7%). Os locais mais frequentes foram a residência (43,8%) e a rua (21,4%). Pacientes negros tiveram maior probabilidade de sofrer agressões (RP=9,0, IC 95%=8,02-10,11; p<0,001) e da ocorrência ser na rua (RP=3,05, IC 95%=2,74-3,39; p<0,001), enquanto pacientes brancos tiveram maior probabilidade de lesões autoprovocadas (RP=10,53, IC 95%=9,39-11,81; p<0,001) e da ocorrência ser na residência (RP=3,64, IC 95%=3,33-3,98; p<0,001). A probabilidade de lesões autoprovocadas aumentou com a idade de forma consistente, atingindo o máximo em pacientes com mais de 80 anos (RP=12,01, IC 95%=7,49-19,23; p<0,001). A pontuação média na escala de coma de Glasgow foi 10 (DP 5,5) e na escala de severidade da lesão foi 18,6 (DP 13,0). A maioria das lesões (53,1%) foi classificada como severa ou muito severa. Dentre os pacientes, 64,6% tiveram lesão cerebral traumática e 16% evoluíram a óbito. Conclusão: A maior parte das lesões oculares relacionadas a armas de fogo ocorreu em pacientes jovens, do sexo masculino, brancos e sulistas. Negros foram afetados desproporcionalmente. A maior parte das lesões oculares relacionadas a armas de fogo apresentou riscos à visão e foi associada a lesões cerebrais traumáticas. A maioria dos pacientes sobreviveu, mas com potencial para invalidez no longo prazo. As diferenças demográficas identificadas podem ser potencialmente alvos de ações preventivas.

Patterns of ocular trauma in elderly patients in an urban population-the Bronx experience / Padrões de trauma ocular em pacientes idosos em uma população urbana - a experiência de Bronx

ABSTRACT Purpose: To evaluate the characteristics of ocular injuries among elderly patients admitted to an urban level I trauma center because of major trauma from 2008 to 2015. Methods: A retrospective chart review was conducted of patients aged >65 years admitted with ocular injuries that were identified with ICD-9 codes. Tabulated data were analyzed using the Student's paired t-test, the chi-squared test, and regression analysis using STATA/MP-12 software. Significance was set at p<0.05. Results: Of a total of 861 patients, 221 (25.7%) admitted for major trauma and ocular injuries were elderly. The mean age of these patients was 80.3 years (median =79.2 years; interquartile range=63.8-94.6 years). Of these patients, 40.7% were males and 59.3% were females. The males were younger than the females (mean age, 77.3 vs. 82.4 years, respectively, p<0.001). Race was documented as white (30.8%), black (13.6%), and "other" (54.3%), with 67.5% of the "other" group (36.7% overall) identified as Hispanic. The most frequent injuries were contusion of the eye/adnexa (68.2%), orbital wall fractures (22.2%), and an open wound of the ocular adnexa (18.1%). Males had a 2.64-fold greater risk of orbital wall fractures (95% confidence interval [CI]=1.38-5.05, p<0.003). Patients with orbital wall fractures had higher injury severity scores than those without (95% CI=14.1-20.9 vs. 6.8-8.6, respectively, p<0.001). The most common injuries were falls (77.8%) and pedestrian/motor vehicle accidents (6.8%). Falls occurred mostly at home (51.7%), on the street (13.9%), and in hospitals/nursing homes (12.2%). Those falling at home were older than those falling at other locations (95% CI=81.8-85.4 vs. 77.0-80.6 years, respectively, p<0.002). Conclusions: Ocular injuries in elderly Bronx patients most commonly occurred in females due to falls in the home/nursing home setting. Public health measures addressing identifiable individual and environmental risks in these common locations would be most beneficial in reducing the incidence of ocular injuries in this population.

RESUMO Objetivo: Avaliar as características das lesões oculares de idosos nas internações por grandes traumatismos em um centro urbano de trauma nível I de 2008 a 2015. Métodos: Realizou-se uma revisão retrospectiva de prontuários de pacientes com mais de 65 anos internados com lesões oculares identificados com os códigos CID-9. Os dados tabulados foram analisados com o teste t de Student, teste qui-quadrado e análise de regressão, utilizando o software STATA/MP-12. A significância estatística foi fixada em p<0,05. Resultados: Duzentos e vinte e um (25,7%) pacientes de um total de 861, admitidos por traumatismo craniano importante e lesões oculares, eram idosos. A idade média era de 80,3 anos (mediana=79,2; intervalo interquartil=63,8-94,6). 40,7% eram do sexo masculino e 59,3% do feminino. Os homens eram menos idosos (média=77,3) do que as mulheres (média=82,4), p<0,001. A raça foi documentada como branca (30,8%), negra (13,6%) e "outra" (54,3%); 67,5% dos "outros" (36,7% no geral) identificados como hispânicos. As lesões mais frequentes foram contusão do olho/anexos (68,2%), fraturas da parede orbital (22,2%) e ferida aberta dos anexos oculares (18,1%). Os homens tiveram 2,64 mais chances de fraturas da parede orbital (95% CI=1,38-5,05; p<0,003). Pacientes com fraturas da parede orbital tiveram maiores escores de gravidade da lesão (95% CI=14,1-20,9) do que aqueles sem fraturas (96% IC=6,8-8,6), p<0,001. Os mecanismos comuns foram quedas (77,8%) e acidentes a pé com veículos automotores (6,8%). As quedas ocorreram principal mente em casa (51,7%), na rua (13,9%) e em hospitais/lares de idosos (12,2%). Aqueles que caíram em casa eram mais velhos (IC 95%=81,8-85,4) do que os que tiveram quedas em outros locais (IC 95%=77,0-80,6), p<0,002. Conclusões: Lesões oculares em pacientes idosos de Bronx foram mais comuns no sexo feminino e devido a quedas que ocorreram em casa/lar de idosos. Medidas de saúde pública direcionadas a riscos individuais e ambientais identificáveis nesses locais comuns seriam mais benéficas na redução de lesões oculares nessa população.

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.

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.

Etiology of optic atrophy: a prospective observational study from Saudi Arabia.

BACKGROUND: Optic atrophy (OA) represents permanent retinal ganglion cell loss warranting study to establish etiology. OBJECTIVES: To describe neurogenic causes of OA. DESIGN: Prospective, observational. SETTING: Tertiary care center, Riyadh, Saudi Arabia. PATIENTS AND METHODS: We included consecutive patients of all ages with OA caused by lesions affecting the visual pathways who were referred over a 9-month period (November 2013 to July 2014). Diagnosis was based on visual acuity, ophthalmoscopic features and ancillary tests. Patient demographics, results of a clinical examination, test data and etiology were recorded. For each cause of OA, both gender and age group were analyzed as potential risk factors using simple univariate logistic regression. OA associated with glaucoma and retinal diseases was excluded. MAIN OUTCOME MEASURE: Description of causes of OA. RESULTS: Two hundred and four patients and 353 eyes met inclusion criteria. The median age was 27 years (range 3 months-77 years; interquartile range, 27 years) among 111(54.4%) females and 93(45.6%) males, with no statistically significant difference in age of presentation between the genders. The majority of lesions were bilateral (n=151, 74%). Tumors were the most common cause, accounting for 127 (62.2%) cases. These occurred mostly in adults (72.4%) compared to the pediatric group (OR=3.3, 95% CI: 1.79-6.03; P < .001). Hereditary neoplasia (OR=5.55; 95% CI: 1.67-18.42; P=.005) and metabolic diseases (OR=17.57; 95% CI: 2.15-143.62; P=.007) were more common causes in the pediatric group. There were no significant associations between gender or visual acuity and etiology of OA. In developed nations, OA is frequently the result of ischemia and neuritis. We found many other causes, especially orbital and intracranial tumors. CONCLUSIONS: The frequency of tumors as the cause of OA may represent a higher incidence of aggressive tumors coupled with poor recognition/acknowledgement of symptoms and limited access, resulting in late presentations. LIMITATIONS: These findings may reflect bias from selective referrals to a tertiary center and may not represent all of Saudi Arabia.

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.

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.

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.

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.

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.

Overexpression of adenosine kinase in cortical astrocytes and focal neocortical epilepsy in mice.

OBJECT: New experimental models and diagnostic methods are needed to better understand the pathophysiology of focal neocortical epilepsies in a search for improved epilepsy treatment options. The authors hypothesized that a focal disruption of adenosine homeostasis in the neocortex might be sufficient to trigger electrographic seizures. They further hypothesized that a focal disruption of adenosine homeostasis might affect microcirculation and thus offer a diagnostic opportunity for the detection of a seizure focus located in the neocortex. METHODS: Focal disruption of adenosine homeostasis was achieved by injecting an adeno-associated virus (AAV) engineered to overexpress adenosine kinase (ADK), the major metabolic clearance enzyme for the brain's endogenous anticonvulsant adenosine, into the neocortex of mice. Eight weeks following virus injection, the affected brain area was imaged via optical microangiography (OMAG) to detect changes in microcirculation. After completion of imaging, cortical electroencephalography (EEG) recordings were obtained from the imaged brain area. RESULTS: Viral expression of the Adk cDNA in astrocytes generated a focal area (~ 2 mm in diameter) of ADK overexpression within the neocortex. OMAG scanning revealed a reduction in vessel density within the affected brain area of approximately 23% and 29% compared with control animals and the contralateral hemisphere, respectively. EEG recordings revealed electrographic seizures within the focal area of ADK overexpression at a rate of 1.3 ± 0.2 seizures per hour (mean ± SEM). CONCLUSIONS: The findings of this study suggest that focal adenosine deficiency is sufficient to generate a neocortical focus of hyperexcitability, which is also characterized by reduced vessel density. The authors conclude that their model constitutes a useful tool to study neocortical epilepsies and that OMAG constitutes a noninvasive diagnostic tool for the imaging of seizure foci with disrupted adenosine homeostasis.

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.

Treatment with low-dose methamphetamine improves behavioral and cognitive function after severe traumatic brain injury.

BACKGROUND: Methamphetamine increases the release and blocks the reuptake of dopamine. The moderate activation of dopamine receptors may elicit neuroprotective effects. We have recently demonstrated that low doses of methamphetamine reduce neuronal loss after ischemic injury. On the basis of this finding, we hypothesized that methamphetamine could also prevent neuronal loss and improve functional behavior after severe traumatic brain injury (TBI). METHODS: The rat lateral fluid percussion injury model was used to generate severe TBI. Three hours after injury, animals were treated with saline or methamphetamine. Neurological severity scores and foot fault assessments were used to determine whether treatment enhanced recovery after injury. The potential for methamphetamine treatment to improve cognitive function was assessed using the Morris water maze. Forty-eight hours after injury, paraffin-embedded brain sections were TUNEL stained to measure apoptotic cell death. Sections were also stained with antibody to doublecortin to quantify immature neurons within the dentate gyrus. RESULTS: Treatment with low-dose methamphetamine significantly reduced both behavioral and cognitive dysfunction after severe TBI. Methamphetamine-treated animals scored significantly lower on neurological severity scores and had significantly less foot faults after TBI compared with saline-treated control rats. Furthermore, methamphetamine treatment restored learning and memory function to near normal ability after TBI. At 48 hours after injury, apoptotic cell death within the hippocampus was significantly reduced, and the presence of immature neurons was significantly increased in methamphetamine-treated rats compared with saline-treated controls. CONCLUSION: Treatment with low-dose methamphetamine after severe TBI elicits a robust neuroprotective response resulting in significant improvements in behavioral and cognitive functions.

Increased NADPH oxidase-derived superoxide is involved in the neuronal cell death induced by hypoxia-ischemia in neonatal hippocampal slice cultures.

Neonatal brain hypoxia-ischemia (HI) results in neuronal cell death. Previous studies indicate that reactive oxygen species, such as superoxide, play a key role in this process. However, the cellular sources have not been established. In this study we examine the role of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex in neonatal HI brain injury and elucidate its mechanism of activation. Rat hippocampal slices were exposed to oxygen glucose deprivation (OGD) to mimic the conditions seen in HI. Initial studies confirmed an important role for NADPH oxidase-derived superoxide in the oxidative stress associated with OGD. Further, the OGD-mediated increase in apoptotic cell death was inhibited by the NADPH oxidase inhibitor apocynin. The activation of NADPH oxidase was found to be dependent on the p38 mitogen-activated protein kinase-mediated phosphorylation and activation of the p47(phox) subunit. Using an adeno-associated virus antisense construct to selectively decrease p47(phox) expression in neurons showed that this led to inhibition of both the increase in superoxide and the neuronal cell death associated with OGD. We also found that NADPH oxidase inhibition in a neonatal rat model of HI or scavenging hydrogen peroxide reduced brain injury. Thus, we conclude that activation of the NADPH oxidase complex contributes to the oxidative stress during HI and that therapies targeted against this complex could provide neuroprotection against the brain injury associated with neonatal HI.

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.

Low dose methamphetamine mediates neuroprotection through a PI3K-AKT pathway.

High doses of methamphetamine induce the excessive release of dopamine resulting in neurotoxicity. However, moderate activation of dopamine receptors can promote neuroprotection. Therefore, we used in vitro and in vivo models of stroke to test the hypothesis that low doses of methamphetamine could induce neuroprotection. We demonstrate that methamphetamine does induce a robust, dose-dependent, neuroprotective response in rat organotypic hippocampal slice cultures exposed to oxygen-glucose deprivation (OGD). A similar dose dependant neuroprotective effect was observed in rats that received an embolic middle cerebral artery occlusion (MCAO). Significant improvements in behavioral outcomes were observed in rats when methamphetamine administration delayed for up to 12 h after MCAO. Methamphetamine-mediated neuroprotection was significantly reduced in slice cultures by the addition of D1 and D2 dopamine receptor antagonist. Treatment of slice cultures with methamphetamine resulted in the dopamine-mediated activation of AKT in a PI3K dependant manner. A similar increase in phosphorylated AKT was observed in the striatum, cortex and hippocampus of methamphetamine treated rats following MCAO. Methamphetamine-mediated neuroprotection was lost in rats when PI3K activity was blocked by wortmannin. Finally, methamphetamine treatment decreased both cleaved caspase 3 levels in slice cultures following OGD and TUNEL staining within the striatum and cortex in rats following transient MCAO. These data indicate that methamphetamine can mediate neuroprotection through activation of a dopamine/PI3K/AKT-signaling pathway.