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1.  Pediatric Super-Refractory Status Epilepticus Treated with Allopregnanolone 
Annals of neurology  2014;76(6):911-915.
Super-refractory status epilepticus is a life-threatening condition. Resistance to benzodiazepine and barbiturate treatment for this disorder is thought to be due to internalization of synaptic γ-aminobutyric acid (GABA)A receptors, and withdrawal of benzodiazepines and barbiturates during treatment often triggers seizure recurrence. The neurosteroid allopregnanolone acts as a positive allosteric modulator of synaptic and extrasynaptic GABAA receptors. Here we describe the use of allopregnanolone in 2 pediatric patients with super-refractory status epilepticus. This treatment allowed the general anesthetic infusions to be weaned with resolution of status epilepticus. This is the first report of allopregnanolone use to treat status epilepticus in children.
PMCID: PMC4534165  PMID: 25363147
2.  Preventable Pediatric Stroke via Vaccination? 
Pediatric Neurology Briefs  null;29(10):76.
Investigators from the Vascular Effects of Infection in Pediatric Stroke (VIPS) group studied the risk of arterial ischemic stroke (AIS) associated with minor infection and routine childhood vaccinations.
PMCID: PMC4747138  PMID: 26933534
Pediatric Stoke; Infection; Vaccination
3.  Herpes simplex virus serotype and entry receptor availability alter CNS disease in a mouse model of neonatal HSV 
Pediatric research  2014;76(6):528-534.
Outcomes of neonates with herpes simplex virus (HSV) encephalitis are worse after infection with HSV-2 when compared with HSV-1. The proteins herpes virus entry mediator (HVEM) and nectin-1 mediate HSV entry into susceptible cells. Prior studies have shown receptor-dependent differences in pathogenesis that depend on route of inoculation and host developmental age.
We investigated serotype-related differences in HSV disease and their relationship to entry receptor availability in a mouse model of encephalitis.
Mortality was attenuated in seven-day-old wild-type (WT) mice inoculated with HSV-1(F) when compared with HSV-2(333). No serotype-specific differences were seen after inoculation of adult mice. HSV-1 pathogenesis was also attenuated relative to HSV-2 in newborn but not adult mice lacking HVEM or nectin-1. HSV-2 requires nectin-1 for encephalitis in adult but not newborn mice; in contrast, nectin-1 was important for HSV-1 pathogenesis in both age groups. Early viral replication was independent of age, viral serotype, or mouse genotype, suggesting host responses influence outcomes. In this regard, significantly greater amounts of inflammatory mediators were detected in brain homogenates from WT newborns 2 days after infection compared with adults and receptor-knockout newborns.
Dysregulation of inflammatory responses induced by infection may influence the severity of HSV encephalitis.
PMCID: PMC4233006  PMID: 25198371
4.  BMP-regulated exosomes from Drosophila male reproductive glands reprogram female behavior 
The Journal of Cell Biology  2014;206(5):671-688.
Male Drosophila reproductive glands secrete exosomes in a BMP-dependent manner that fuse with sperm after mating and suppress female remating.
Male reproductive glands secrete signals into seminal fluid to facilitate reproductive success. In Drosophila melanogaster, these signals are generated by a variety of seminal peptides, many produced by the accessory glands (AGs). One epithelial cell type in the adult male AGs, the secondary cell (SC), grows selectively in response to bone morphogenetic protein (BMP) signaling. This signaling is involved in blocking the rapid remating of mated females, which contributes to the reproductive advantage of the first male to mate. In this paper, we show that SCs secrete exosomes, membrane-bound vesicles generated inside late endosomal multivesicular bodies (MVBs). After mating, exosomes fuse with sperm (as also seen in vitro for human prostate-derived exosomes and sperm) and interact with female reproductive tract epithelia. Exosome release was required to inhibit female remating behavior, suggesting that exosomes are downstream effectors of BMP signaling. Indeed, when BMP signaling was reduced in SCs, vesicles were still formed in MVBs but not secreted as exosomes. These results demonstrate a new function for the MVB–exosome pathway in the reproductive tract that appears to be conserved across evolution.
PMCID: PMC4151142  PMID: 25154396
5.  Childhood Stroke and Congenital Heart Disease 
Pediatric Neurology Briefs  null;29(3):18.
Investigators from the University of California performed a case-control study of the association of stroke with congenital heart disease (CHD) within a population of 2.5 million children in Northern California.
PMCID: PMC4747257  PMID: 26933560
Epidemiology; Stroke; Pediatrics; Congenital Heart Disease
6.  Continuous EEG in Critically Ill Children 
Pediatric Neurology Briefs  null;29(3):20.
Investigators from the Critical Care Continuous EEG Task Force of the American Clinical Neurophysiology Society reported a consensus statement on indications for the use of critical care continuous electroencephalographic monitoring (ccEEG) in adults and children.
PMCID: PMC4747259  PMID: 26933562
EEG; Critical Care; Pediatrics
7.  Acute Care Clinical Indicators Associated with Discharge Outcomes in Children with Severe Traumatic Brain Injury 
Critical care medicine  2014;42(10):2258-2266.
The relationship between acute care clinical indicators in the first severe Pediatric traumatic brain injury (TBI) Guidelines and outcomes have not been examined. We aimed to develop a set of acute care guideline-influenced clinical indicators of adherence and tested the relationship between these indicators during the first 72 hours after hospital admission and discharge outcomes.
Retrospective multicenter cohort study
Five regional pediatric trauma centers affiliated with academic medical centers.
Children under 17 years with severe TBI (admission Glasgow coma scale (GCS) score ≤ 8, ICD-9 diagnosis codes of 800.0-801.9, 803.0-804.9, 850.0-854.1, 959.01, 950.1-950.3, 995.55, maximum head abbreviated injury severity score ≥ 3) who received tracheal intubation for at-least 48 hours in the intensive care unit (ICU) between 2007 -2011 were examined.
Measurements and Main Results
Total percent adherence to the clinical indicators across all treatment locations (pre-hospital [PH], emergency department [ED], operating room [OR], and intensive care unit [ICU]) during the first 72 hours after admission to study center were determined. Main outcomes were discharge survival and Glasgow outcome scale (GOS) score.
Total adherence rate across all locations and all centers ranged from 68-78%. Clinical indicators of adherence were associated with survival (aHR 0.94; 95 % CI 0.91, 0.96). Three indicators were associated with survival: absence of PH hypoxia (aHR 0.20; 95% CI 0.08, 0.46), early ICU start of nutrition (aHR 0.06; 95% CI 0.01, 0.26), and ICU PaCO2 >30 mm Hg in the absence of radiographic or clinical signs of cerebral herniation (aHR 0.22; 95% CI 0.06, 0.8). Clinical indicators of adherence were associated with favorable GOS among survivors, (aHR 0.99; 95% CI 0.98, 0.99). Three indicators were associated with favorable discharge GOS: all OR CPP >40 mm Hg (aRR 0.64; 95% CI 0.55, 0.75), all ICU CPP > 40mm Hg (aRR 0.74; 95% CI 0.63, 0.87), and no surgery (any type; aRR 0.72; 95% CI 0.53, 0.97).
Acute care clinical indicators of adherence to the Pediatric Guidelines were associated with significantly higher discharge survival and improved discharge GOS. Some indicators were protective, regardless of treatment location, suggesting the need for an interdisciplinary approach to the care of children with severe TBI.
PMCID: PMC4167478  PMID: 25083982
pediatrics; trauma; brain injury; indicators; outcomes; injury
8.  In Vitro Photodynamic Inactivation of Plant-Pathogenic Fungi Colletotrichum acutatum and Colletotrichum gloeosporioides with Novel Phenothiazinium Photosensitizers 
The increasing tolerance to currently used fungicides in both clinical and agricultural areas is of great concern. The nonconventional light-based approach of antimicrobial photodynamic treatment (APDT) is a promising alternative to conventional fungicides. We evaluated the effects of APDT with four phenothiazinium derivatives (methylene blue [MB], new methylene blue N [NMBN], toluidine blue O [TBO], and the novel pentacyclic phenothiazinium photosensitizer [PS] S137) on conidia of three fungal species (Colletotrichum acutatum, Colletotrichum gloeosporioides, and Aspergillus nidulans). The efficacy of APDT with each PS was determined, initially, based on photosensitizer MICs. Additionally, the effects of APDT with two selected PSs (NMBN and S137) on survival of conidia were evaluated. The subcellular localization of the PS in C. acutatum conidia was determined. The effects of photodynamic treatments on leaves of the plant host Citrus sinensis were also investigated. APDT with S137 showed the lowest MIC. MICs for S137 were 5 μM for the three fungal species when a fluence of 25 J cm−2 was used. APDT with NMBN (50 μM) and S137 (10 μM) resulted in a reduction in the survival of the conidia of all species of approximately 5 logs with fluences of ≥15 J cm−2. Washing of the conidia before light exposure did not prevent photodynamic inactivation. Both NMBN and S137 accumulated in cytoplasmic structures, such as lipid bodies, of C. acutatum conidia. No damage to orange tree leaves was observed after APDT.
PMCID: PMC3957600  PMID: 24362436
9.  National Variability in Intracranial Pressure Monitoring and Craniotomy for Children With Moderate to Severe Traumatic Brain Injury 
Neurosurgery  2013;73(5):746-752.
Traumatic brain injury (TBI) is a significant cause of mortality and disability in children. Intracranial pressure monitoring (ICPM) and craniotomy/craniectomy (CRANI) may affect outcomes. Sources of variability in the use of these interventions remain incompletely understood.
To analyze sources of variability in the use of ICPM and CRANI.
Retrospective cross-sectional study of patients with moderate/severe pediatric TBI with the use of data submitted to the American College of Surgeons National Trauma Databank.
We analyzed data from 7140 children at 156 US hospitals during 7 continuous years. Of the children, 27.4% had ICPM, whereas 11.7% had a CRANI. Infants had lower rates of ICPM and CRANI than older children. A lower rate of ICPM was observed among children hospitalized at combined pediatric/adult trauma centers than among children treated at adult-only trauma centers (relative risk = 0.80; 95% confidence interval 0.66-0.97). For ICPM and CRANI, 18.5% and 11.6%, respectively, of residual model variance was explained by between-hospital variation in care delivery, but almost no correlation was observed between within-hospital tendency toward performing these procedures.
Infants received less ICPM than older children, and children hospitalized at pediatric trauma centers received less ICPM than children at adult-only trauma centers. In addition, significant between-hospital variability existed in the delivery of ICPM and CRANI to children with moderate-severe TBI.
PMCID: PMC4127400  PMID: 23863766
Decompressive craniectomy; Intracranial pressure monitoring
10.  Triage of Children with Moderate and Severe Traumatic Brain Injury to Trauma Centers 
Journal of Neurotrauma  2013;30(13):1129-1136.
Outcomes after pediatric traumatic brain injury (TBI) are related to pre-treatment factors including age, injury severity, and mechanism of injury, and may be positively affected by treatment at trauma centers relative to non-trauma centers. This study estimated the proportion of children with moderate to severe TBI who receive care at trauma centers, and examined factors associated with receipt of care at adult (ATC), pediatric (PTC), and adult/pediatric trauma centers (APTC), compared with care at non-trauma centers (NTC) using a nationally representative database. The Kids' Inpatient Database was used to identify hospitalizations for moderate to severe pediatric TBI. Pediatric inpatients ages 0 to 17 years with at least one diagnosis of TBI and a maximum head Abbreviated Injury Scale score of ≥3 were studied. Multinomial logistic regression was performed to examine factors predictive of the level and type of facility where care was received. A total of 16.7% of patients were hospitalized at NTC, 44.2% at Level I or II ATC, 17.9% at Level I or II PTC, and 21.2% at Level I or II APTC. Multiple regression analyses showed receipt of care at a trauma center was associated with age and polytrauma. We concluded that almost 84% of children with moderate to severe TBI currently receive care at a Level I or Level II trauma center. Children with trauma to multiple body regions in addition to more severe TBI are more likely to receive care a trauma center relative to a NTC.
PMCID: PMC3700462  PMID: 23343131
brain injury; pediatrics; trauma; trauma center
11.  The Temporal Version of the Pediatric Sepsis Biomarker Risk Model 
PLoS ONE  2014;9(3):e92121.
PERSEVERE is a risk model for estimating mortality probability in pediatric septic shock, using five biomarkers measured within 24 hours of clinical presentation.
Here, we derive and test a temporal version of PERSEVERE (tPERSEVERE) that considers biomarker values at the first and third day following presentation to estimate the probability of a “complicated course”, defined as persistence of ≥2 organ failures at seven days after meeting criteria for septic shock, or death within 28 days.
Biomarkers were measured in the derivation cohort (n = 225) using serum samples obtained during days 1 and 3 of septic shock. Classification and Regression Tree (CART) analysis was used to derive a model to estimate the risk of a complicated course. The derived model was validated in the test cohort (n = 74), and subsequently updated using the combined derivation and test cohorts.
A complicated course occurred in 23% of the derivation cohort subjects. The derived model had a sensitivity for a complicated course of 90% (95% CI 78–96), specificity was 70% (62–77), positive predictive value was 47% (37–58), and negative predictive value was 96% (91–99). The area under the receiver operating characteristic curve was 0.85 (0.79–0.90). Similar test characteristics were observed in the test cohort. The updated model had a sensitivity of 91% (81–96), a specificity of 70% (64–76), a positive predictive value of 47% (39–56), and a negative predictive value of 96% (92–99).
tPERSEVERE reasonably estimates the probability of a complicated course in children with septic shock. tPERSEVERE could potentially serve as an adjunct to physiological assessments for monitoring how risk for poor outcomes changes during early interventions in pediatric septic shock.
PMCID: PMC3953585  PMID: 24626215
12.  A Genetic Screen Based on in Vivo RNA Imaging Reveals Centrosome-Independent Mechanisms for Localizing gurken Transcripts in Drosophila 
G3: Genes|Genomes|Genetics  2014;4(4):749-760.
We have screened chromosome arm 3L for ethyl methanesulfonate−induced mutations that disrupt localization of fluorescently labeled gurken (grk) messenger (m)RNA, whose transport along microtubules establishes both major body axes of the developing Drosophila oocyte. Rapid identification of causative mutations by single-nucleotide polymorphism recombinational mapping and whole-genomic sequencing allowed us to define nine complementation groups affecting grk mRNA localization and other aspects of oogenesis, including alleles of elg1, scaf6, quemao, nudE, Tsc2/gigas, rasp, and Chd5/Wrb, and several null alleles of the armitage Piwi-pathway gene. Analysis of a newly induced kinesin light chain allele shows that kinesin motor activity is required for both efficient grk mRNA localization and oocyte centrosome integrity. We also show that initiation of the dorsoanterior localization of grk mRNA precedes centrosome localization, suggesting that microtubule self-organization contributes to breaking axial symmetry to generate a unique dorsoventral axis.
PMCID: PMC4059244  PMID: 24531791
oogenesis; RNA localization; microtubules; whole-genome sequencing; mutant mapping
13.  Testing the Prognostic Accuracy of the Updated Pediatric Sepsis Biomarker Risk Model 
PLoS ONE  2014;9(1):e86242.
We previously derived and validated a risk model to estimate mortality probability in children with septic shock (PERSEVERE; PEdiatRic SEpsis biomarkEr Risk modEl). PERSEVERE uses five biomarkers and age to estimate mortality probability. After the initial derivation and validation of PERSEVERE, we combined the derivation and validation cohorts (n = 355) and updated PERSEVERE. An important step in the development of updated risk models is to test their accuracy using an independent test cohort.
To test the prognostic accuracy of the updated version PERSEVERE in an independent test cohort.
Study subjects were recruited from multiple pediatric intensive care units in the United States. Biomarkers were measured in 182 pediatric subjects with septic shock using serum samples obtained during the first 24 hours of presentation. The accuracy of PERSEVERE 28-day mortality risk estimate was tested using diagnostic test statistics, and the net reclassification improvement (NRI) was used to test whether PERSEVERE adds information to a physiology-based scoring system.
Mortality in the test cohort was 13.2%. Using a risk cut-off of 2.5%, the sensitivity of PERSEVERE for mortality was 83% (95% CI 62–95), specificity was 75% (68–82), positive predictive value was 34% (22–47), and negative predictive value was 97% (91–99). The area under the receiver operating characteristic curve was 0.81 (0.70–0.92). The false positive subjects had a greater degree of organ failure burden and longer intensive care unit length of stay, compared to the true negative subjects. When adding PERSEVERE to a physiology-based scoring system, the net reclassification improvement was 0.91 (0.47–1.35; p<0.001).
The updated version of PERSEVERE estimates mortality probability reliably in a heterogeneous test cohort of children with septic shock and provides information over and above a physiology-based scoring system.
PMCID: PMC3906040  PMID: 24489704
14.  Selective head cooling for the treatment of neurologic complications of acute liver failure in a newborn with disseminated herpes infection 
SpringerPlus  2013;2:572.
Neurologic complications of pediatric acute liver failure (ALF) are a major determinant of outcome. Management of these complications, including increased intracranial pressure (ICP) is largely supportive. Although hypothermia is an effective treatment for perinatal asphyxia and is used to reduce ICP following traumatic brain injury, it has not been evaluated for neurologic complications of ALF in the newborn.
Case report.
We present a case of neonatal herpes simplex virus (HSV)-associated ALF with profound neurologic impairment and increased ICP. The patient was treated with selective head cooling, and monitored with transcranial doppler (TCD) studies of cerebral blood flow velocity, and electroencephalograms (EEG). The duration of head cooling was influenced by absent diastolic flow on TCDs, which subsequently improved during hypothermia. Continuous EEGs captured subclinical seizures, which improved with antiepileptic medications. Her death was attributed to a massive pulmonary hemorrhage and a hypoxemic cardiac arrest secondary to significant coagulopathy.
This case demonstrates that selective head cooling may attenuate increased ICP in neonatal encephalopathy, and that TCDs may guide management in the absence of invasive monitoring.
PMCID: PMC3825224  PMID: 24255865
Acute liver failure; Transcranial doppler; EEG; Neonate; Hypothermia
Free radical biology & medicine  2012;53(5):1139-1151.
Neonatal brain hypoxia ischemia (HI) results in neuronal cell death. Previous studies indicate that reactive oxygen species (ROS) such as superoxide, play a key role in this process. However, the cellular sources have not been established. In this study we examined the role of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex in neonatal HI brain injury and elucidated 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 p47phox subunit. Using an adeno-associated virus antisense construct to selectively decrease p47phox expression in neurons, and showed that this lead to inhibition both of the increase in superoxide and neuronal cell death associated with OGD. We also found that NADPH oxidase inhibition in a neonatal rat model of HI or scavenging hydrogen peroxide (H2O2) 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 exhibit neuroprotection against the brain injury associated with neonatal HI.
PMCID: PMC3527086  PMID: 22728269
Hypoxia-ischemia; neuronal cell death; apoptosis; superoxide; Hydrogen peroxide; NADPH oxidase; p47phox; p38MAP kinase; neonatal brain
16.  Evaluation of Asymmetric Dimethylarginine, Arginine, and Carnitine Metabolism in Pediatric Sepsis 
Pediatric Critical Care Medicine  2012;13(4):e210-e218.
Increased plasma concentrations of the endogenous nitric oxide (NO) synthase inhibitor, asymmetric dimethylarginine (ADMA), decreased arginine bioavailability, and mitochondrial dysfunction have been reported in adult sepsis. We studied whether ADMA, arginine, and carnitine metabolism (a measure of mitochondrial dysfunction) are altered in pediatric sepsis and whether these are clinically useful biomarkers.
Prospective, observational study
Pediatric intensive care unit at an academic medical center
Ninety patients ≤ 18 years-old—30 with severe sepsis or septic shock compared with thirty age-matched febrile and thirty age-matched healthy controls.
Measurements and Main Results
Plasma ADMA and whole blood arginine, citrulline, ornithine, and acylcarnitine:free carnitine (AC:FC) ratio were measured daily for septic patients and once for controls using tandem mass spectrometry. Plasma ADMA concentration (median, IQR µmol/L) on day 1 was lower in severe sepsis and septic shock (0.38, 0.30–0.56) compared with febrile (0.45, 0.40–0.59) and healthy (0.60, 0.54–0.67) controls (p<0.001), though decreased ADMA was predominantly found in neutropenic patients. Day 1 arginine was lower in septic (10, IQR 7–20 µmol/L) compared with healthy patients (32, IQR 23–40; p<0.001), and the arginine:ornithine ratio was decreased in sepsis, indicating increased arginase activity (an alternative pathway for arginine metabolism). The arginine:ADMA and AC:FC ratios did not differ between septic and control patients. ADMA was inversely correlated with organ dysfunction by PELOD score (r=−0.50, p=0.009), interleukin-6 (r=−0.55, p=0.01), and interleukin-8 (r=−0.52, p=0.03) on admission. Arginine, arginine:ADMA, and AC:FC were not associated with organ dysfunction or outcomes.
ADMA was decreased in pediatric sepsis and was inversely associated with inflammation and organ dysfunction. This suggests that inhibition of NO synthase by ADMA accumulation is unlikely to impact sepsis pathophysiology in septic children despite decreased arginine bioavailability. We did not find an association of ADMA with altered carnitine metabolism, nor were ADMA, arginine, and AC:FC useful as clinical biomarkers.
PMCID: PMC3392424  PMID: 22460770
Nitric oxide; nitric oxide synthase; arginine; carnitine; sepsis; intensive care units, pediatric
17.  Photobactericides—A Local Option against Multi-Drug Resistant Bacteria 
Antibiotics  2013;2(2):182-190.
The use of light-activated bactericidal agents—photobactericides—is suggested in local infection in order to conserve conventional antibacterials for more systemic disease. Local administration of a photobactericide such as methylene blue coupled with locally-targeted red light illumination ensures the production of non-specific reactive oxygen species and thus a rapid and localised antibacterial response, regardless of the conventional resistance status. To this end, the response of photobactericides to conventional resistance mechanisms, and their potential use in infection, is discussed.
PMCID: PMC4790333
photobactericide; conventional resistance mechanisms; methylene blue
18.  Common Data Elements for Pediatric Traumatic Brain Injury: Recommendations from the Working Group on Demographics and Clinical Assessment 
Journal of Neurotrauma  2012;29(4):639-653.
The Common Data Elements (CDEs) initiative is a National Institutes of Health (NIH) interagency effort to standardize naming, definitions, and data structure for clinical research variables. Comparisons of the results of clinical studies of neurological disorders have been hampered by variability in data coding, definitions, and procedures for sample collection. The CDE project objective is to enable comparison of future clinical trials results in major neurological disorders, including traumatic brain injury (TBI), stroke, multiple sclerosis, and epilepsy. As part of this effort, recommendations for CDEs for research on TBI were developed through a 2009 multi-agency initiative. Following the initial recommendations of the Working Group on Demographics and Clinical Assessment, a separate workgroup developed recommendations on the coding of clinical and demographic variables specific to pediatric TBI studies for subjects younger than 18 years. This article summarizes the selection of measures by the Pediatric TBI Demographics and Clinical Assessment Working Group. The variables are grouped into modules which are grouped into categories. For consistency with other CDE working groups, each variable was classified by priority (core, supplemental, and emerging). Templates were produced to summarize coding formats, guide selection of data points, and provide procedural recommendations. This proposed standardization, together with the products of the other pediatric TBI working groups in imaging, biomarkers, and outcome assessment, will facilitate multi-center studies, comparison of results across studies, and high-quality meta-analyses of individual patient data.
PMCID: PMC3289844  PMID: 21939389
clinical studies; common data elements; data coding; data collection; pediatric; standardization; traumatic brain injury
19.  Oxygen Glucose Deprivation in Rat Hippocampal Slice Cultures Results in Alterations in Carnitine Homeostasis and Mitochondrial Dysfunction 
PLoS ONE  2012;7(9):e40881.
Mitochondrial dysfunction characterized by depolarization of mitochondrial membranes and the initiation of mitochondrial-mediated apoptosis are pathological responses to hypoxia-ischemia (HI) in the neonatal brain. Carnitine metabolism directly supports mitochondrial metabolism by shuttling long chain fatty acids across the inner mitochondrial membrane for beta-oxidation. Our previous studies have shown that HI disrupts carnitine homeostasis in neonatal rats and that L-carnitine can be neuroprotective. Thus, this study was undertaken to elucidate the molecular mechanisms by which HI alters carnitine metabolism and to begin to elucidate the mechanism underlying the neuroprotective effect of L-carnitine (LCAR) supplementation. Utilizing neonatal rat hippocampal slice cultures we found that oxygen glucose deprivation (OGD) decreased the levels of free carnitines (FC) and increased the acylcarnitine (AC): FC ratio. These changes in carnitine homeostasis correlated with decreases in the protein levels of carnitine palmitoyl transferase (CPT) 1 and 2. LCAR supplementation prevented the decrease in CPT1 and CPT2, enhanced both FC and the AC∶FC ratio and increased slice culture metabolic viability, the mitochondrial membrane potential prior to OGD and prevented the subsequent loss of neurons during later stages of reperfusion through a reduction in apoptotic cell death. Finally, we found that LCAR supplementation preserved the structural integrity and synaptic transmission within the hippocampus after OGD. Thus, we conclude that LCAR supplementation preserves the key enzymes responsible for maintaining carnitine homeostasis and preserves both cell viability and synaptic transmission after OGD.
PMCID: PMC3439445  PMID: 22984394
20.  Antimicrobial photodynamic therapy in the colon: delivering a light punch to the guts? 
Photochemistry and photobiology  2011;87(4):754-756.
A paper in this issue of Photochemistry and Photobiology by Cassidy et al describes the use of a sophisticated drug delivery vehicle prepared by the hot melt extrusion process to deliver photosensitizers to the colon. The smart vehicle protects its cargo through the acidic environment of the stomach but releases the active photosensitizers in the higher pH and anaerobic environment of the colon. The goal is to use photodynamic therapy (PDT) to destroy pathogenic microorganisms that can cause disease when they grow out of control in the colon. Since the colon is an environment with a low oxygen concentration the investigators also used tetrachlorodecaoxide, an oxygen donor to boost the available oxygen concentration. The paper reports results with Enterococcus faecalis and Bacteroides fragilis but the real medical problem demanding to be solved is Clostridium difficile that can cause intractable drug-resistant infections after antibiotic use. There still remain barriers to implementing this strategy in vivo, including light delivery to the upper colon, oxygen availability and optimizing the selectivity of photosensitizers for bacteria over colon epithelial cells. Nevertheless this highly innovative paper lays the ground for the study of an entirely new and significant application for antimicrobial PDT.
PMCID: PMC3132272  PMID: 21418077
Journal of neuroscience research  2011;89(6):852-861.
Myosin light chain kinase (MLCK) plays an important role in the reorganization of the cytoskeleton leading to disruption of vascular barrier integrity in multiple organs including the blood brain barrier (BBB) after traumatic brain injury (TBI). MLCK has been linked to transforming growth factor (TGF) and rho kinase signaling pathways, but the mechanisms regulating MLCK expression following TBI are not well understood. Albumin leaks into the brain parenchyma following TBI, activates glia and has been linked to TGF-β receptor signaling. We investigated the role of albumin in the increase in MLCK in astrocytes and the signaling pathways involved in this increase. Following midline closed-skull TBI in mice, there was a significant increase in MLCK-immunoreactive (IR) cells and albumin extravasation, which was prevented by treatment with the MLCK inhibitor ML-7. Using immunohistochemical methods, we identified the MCLK-IR cells as astrocytes. In primary astrocytes, exposure to albumin increased both isoforms of MLCK, 130 and 210. Inhibition of the TGF-β receptor partially prevented the albumin-induced increase in both isoforms, which was not prevented by inhibition of smad3. Inhibition of p38 MAPK, but not ERK, JNK or rho kinase also prevented this increase. These results are further evidence of a role of MCLK in the mechanisms of BBB compromise following TBI, and identify astrocytes as a cell type, in addition to endothelium in the BBB which express MLCK. These findings implicate albumin, acting through p38 MAPK, in a novel mechanism by which activation of MLCK following TBI may lead to compromise of the BBB.
PMCID: PMC3079319  PMID: 21360574
myosin light chain kinase; blood brain barrier; astrocyte; traumatic brain injury; transforming growth factor
22.  Albumin induces upregulation of matrix metalloproteinase-9 in astrocytes via MAPK and reactive oxygen species-dependent pathways 
Astrocytes are an integral component of the blood–brain barrier (BBB) which may be compromised by ischemic or traumatic brain injury. In response to trauma, astrocytes increase expression of the endopeptidase matrix metalloproteinase (MMP)-9. Compromise of the BBB leads to the infiltration of fluid and blood-derived proteins including albumin into the brain parenchyma. Albumin has been previously shown to activate astrocytes and induce the production of inflammatory mediators. The effect of albumin on MMP-9 activation in astrocytes is not known. We investigated the molecular mechanisms underlying the production of MMP-9 by albumin in astrocytes.
Primary enriched astrocyte cultures were used to investigate the effects of exposure to albumin on the release of MMP-9. MMP-9 expression was analyzed by zymography. The involvement of mitogen-activated protein kinase (MAPK), reactive oxygen species (ROS) and the TGF-β receptor-dependent pathways were investigated using pharmacological inhibitors. The production of ROS was observed by dichlorodihydrofluorescein diacetate fluorescence. The level of the MMP-9 inhibitor tissue inhibitor of metalloproteinase (TIMP)-1 produced by astrocytes was measured by ELISA.
We found that albumin induces a time-dependent release of MMP-9 via the activation of p38 MAPK and extracellular signal regulated kinase, but not Jun kinase. Albumin-induced MMP-9 production also involves ROS production upstream of the MAPK pathways. However, albumin-induced increase in MMP-9 is independent of the TGF-β receptor, previously described as a receptor for albumin. Albumin also induces an increase in TIMP-1 via an undetermined mechanism.
These results link albumin (acting through ROS and the p38 MAPK) to the activation of MMP-9 in astrocytes. Numerous studies identify a role for MMP-9 in the mechanisms of compromise of the BBB, epileptogenesis, or synaptic remodeling after ischemia or traumatic brain injury. The increase in MMP-9 produced by albumin further implicates both astrocytes and albumin in the acute and long-term complications of acute CNS insults, including cerebral edema and epilepsy.
PMCID: PMC3419618  PMID: 22507553
Matrix metalloproteinase; Astrocyte; Blood brain barrier; Mitogen-activated protein kinases
23.  Pilot Study of the Association of the DDAH2 −449G Polymorphism with Asymmetric Dimethylarginine and Hemodynamic Shock in Pediatric Sepsis 
PLoS ONE  2012;7(3):e33355.
Genetic variability in the regulation of the nitric oxide (NO) pathway may influence hemodynamic changes in pediatric sepsis. We sought to determine whether functional polymorphisms in DDAH2, which metabolizes the NO synthase inhibitor asymmetric dimethylarginine (ADMA), are associated with susceptibility to sepsis, plasma ADMA, distinct hemodynamic states, and vasopressor requirements in pediatric septic shock.
Methodology/Principal Findings
In a prospective study, blood and buccal swabs were obtained from 82 patients ≤18 years (29 with severe sepsis/septic shock plus 27 febrile and 26 healthy controls). Plasma ADMA was measured using tandem mass spectrometry. DDAH2 gene was partially sequenced to determine the −871 6g/7g insertion/deletion and −449G/C single nucleotide polymorphisms. Shock type (“warm” versus “cold”) was characterized by clinical assessment. The −871 7g allele was more common in septic (17%) then febrile (4%) and healthy (8%) patients, though this was not significant after controlling for sex and race (p = 0.96). ADMA did not differ between −871 6g/7g genotypes. While genotype frequencies also did not vary between groups for the −449G/C SNP (p = 0.75), septic patients with at least one −449G allele had lower ADMA (median, IQR 0.36, 0.30–0.41 µmol/L) than patients with the −449CC genotype (0.55, 0.49–0.64 µmol/L, p = 0.008) and exhibited a higher incidence of “cold” shock (45% versus 0%, p = 0.01). However, after controlling for race, the association with shock type became non-significant (p = 0.32). Neither polymorphism was associated with inotrope score or vasoactive infusion duration.
The −449G polymorphism in the DDAH2 gene was associated with both low plasma ADMA and an increased likelihood of presenting with “cold” shock in pediatric sepsis, but not with vasopressor requirement. Race, however, was an important confounder. These results support and justify the need for larger studies in racially homogenous populations to further examine whether genotypic differences in NO metabolism contribute to phenotypic variability in sepsis pathophysiology.
PMCID: PMC3299781  PMID: 22428028
24.  Standardizing Data Collection in Traumatic Brain Injury 
Journal of Neurotrauma  2011;28(2):177-187.
Collaboration among investigators, centers, countries, and disciplines is essential to advancing the care for traumatic brain injury (TBI). It is thus important that we “speak the same language.” Great variability, however, exists in data collection and coding of variables in TBI studies, confounding comparisons between and analysis across different studies. Randomized controlled trials can never address the many uncertainties concerning treatment approaches in TBI. Pooling data from different clinical studies and high-quality observational studies combined with comparative effectiveness research may provide excellent alternatives in a cost-efficient way. Standardization of data collection and coding is essential to this end. Common data elements (CDEs) are presented for demographics and clinical variables applicable across the broad spectrum of TBI. Most recommendations represent a consensus derived from clinical practice. Some recommendations concern novel approaches, for example assessment of the intensity of therapy in severely injured patients. Up to three levels of detail for coding data elements were developed: basic, intermediate, and advanced, with the greatest level of detail attained in the advanced version. More detailed codings can be collapsed into the basic version. Templates were produced to summarize coding formats, explanation of choices, and recommendations for procedures. Endorsement of the recommendations has been obtained from many authoritative organizations. The development of CDEs for TBI should be viewed as a continuing process; as more experience is gained, refinement and amendments will be required. This proposed process of standardization will facilitate comparative effectiveness research and encourage high-quality meta-analysis of individual patient data.
PMCID: PMC3037806  PMID: 21162610
clinical studies; common data elements; data coding; data collection; standardization; traumatic brain injury
25.  Minozac Treatment Prevents Increased Seizure Susceptibility in a Mouse “Two-Hit” Model of Closed Skull Traumatic Brain Injury and Electroconvulsive Shock-Induced Seizures 
Journal of Neurotrauma  2010;27(7):1283-1295.
The mechanisms linking traumatic brain injury (TBI) to post-traumatic epilepsy (PTE) are not known and no therapy for prevention of PTE is available. We used a mouse closed-skull midline impact model to test the hypotheses that TBI increases susceptibility to seizures in a “two-hit” injury model, and that suppression of cytokine upregulation after the first hit will attenuate the increased susceptibility to the second neurological insult. Adult male CD-1 mice underwent midline closed skull pneumatic impact. At 3 and 6 h after impact or sham procedure, the mice were injected IP with either Minozac (Mzc), a suppressor of proinflammatory cytokine upregulation, or vehicle (saline). On day 7 after sham operation or TBI, seizures were induced using electroconvulsive shock (ECS), and susceptibility to seizures was measured by the current required for seizure induction. Activation of glia, neuronal injury, and metallothionein-immunoreactive cells were quantified in the hippocampus by immunohistochemical methods. Neurobehavioral function over 14-day recovery was quantified using the Barnes maze. Following TBI there was a significant increase in susceptibility to seizures induced by ECS, and this susceptibility was prevented by suppression of cytokine upregulation with Mzc. Astrocyte activation, metallothionein expression, and neurobehavioral impairment were also increased in the two-hit group subjected to combined TBI and ECS. These enhanced responses in the two-hit group were also prevented by suppression of proinflammatory cytokine upregulation with Mzc. These data implicate glial activation in the mechanisms of epileptogenesis after TBI, and identify a potential therapeutic approach to attenuate the delayed neurological sequelae of TBI.
PMCID: PMC2942875  PMID: 20486807
astrocyte; electroconvulsive shock; microglia; seizure; traumatic brain injury

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