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1.  The Temporal Version of the Pediatric Sepsis Biomarker Risk Model 
PLoS ONE  2014;9(3):e92121.
Background
PERSEVERE is a risk model for estimating mortality probability in pediatric septic shock, using five biomarkers measured within 24 hours of clinical presentation.
Objective
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.
Methods
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.
Results
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).
Conclusions
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.
doi:10.1371/journal.pone.0092121
PMCID: PMC3953585  PMID: 24626215
2.  Testing the Prognostic Accuracy of the Updated Pediatric Sepsis Biomarker Risk Model 
PLoS ONE  2014;9(1):e86242.
Background
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.
Objective
To test the prognostic accuracy of the updated version PERSEVERE in an independent test cohort.
Methods
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.
Results
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).
Conclusions
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.
doi:10.1371/journal.pone.0086242
PMCID: PMC3906040  PMID: 24489704
3.  Selective head cooling for the treatment of neurologic complications of acute liver failure in a newborn with disseminated herpes infection 
SpringerPlus  2013;2:572.
Background
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.
Methods
Case report.
Results
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.
Conclusion
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.
doi:10.1186/2193-1801-2-572
PMCID: PMC3825224  PMID: 24255865
Acute liver failure; Transcranial doppler; EEG; Neonate; Hypothermia
4.  INCREASED NADPH OXIDASE DERIVED SUPEROXIDE IS INVOLVED IN THE NEURONAL CELL DEATH INDUCED BY HYPOXIA ISCHEMIA IN NEONATAL HIPPOCAMPAL SLICE CULTURES 
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.
doi:10.1016/j.freeradbiomed.2012.06.012
PMCID: PMC3527086  PMID: 22728269
Hypoxia-ischemia; neuronal cell death; apoptosis; superoxide; Hydrogen peroxide; NADPH oxidase; p47phox; p38MAP kinase; neonatal brain
5.  Evaluation of Asymmetric Dimethylarginine, Arginine, and Carnitine Metabolism in Pediatric Sepsis 
Pediatric Critical Care Medicine  2012;13(4):e210-e218.
Objective
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.
Design
Prospective, observational study
Setting
Pediatric intensive care unit at an academic medical center
Patients
Ninety patients ≤ 18 years-old—30 with severe sepsis or septic shock compared with thirty age-matched febrile and thirty age-matched healthy controls.
Interventions
None.
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.
Conclusions
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.
doi:10.1097/PCC.0b013e318238b5cd
PMCID: PMC3392424  PMID: 22460770
Nitric oxide; nitric oxide synthase; arginine; carnitine; sepsis; intensive care units, pediatric
6.  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.
Abstract
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.
doi:10.1089/neu.2011.1952
PMCID: PMC3289844  PMID: 21939389
clinical studies; common data elements; data coding; data collection; pediatric; standardization; traumatic brain injury
7.  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.
doi:10.1371/journal.pone.0040881
PMCID: PMC3439445  PMID: 22984394
8.  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.
doi:10.1111/j.1751-1097.2011.00925.x
PMCID: PMC3132272  PMID: 21418077
9.  ALBUMIN CAUSES INCREASED MYOSIN LIGHT CHAIN KINASE EXPRESSION IN ASTROCYTES VIA P38 MITOGEN ACTIVATED PROTEIN KINASE 
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.
doi:10.1002/jnr.22600
PMCID: PMC3079319  PMID: 21360574
myosin light chain kinase; blood brain barrier; astrocyte; traumatic brain injury; transforming growth factor
10.  Albumin induces upregulation of matrix metalloproteinase-9 in astrocytes via MAPK and reactive oxygen species-dependent pathways 
Background
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1186/1742-2094-9-68
PMCID: PMC3419618  PMID: 22507553
Matrix metalloproteinase; Astrocyte; Blood brain barrier; Mitogen-activated protein kinases
11.  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.
Background
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.
Conclusions/Significance
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.
doi:10.1371/journal.pone.0033355
PMCID: PMC3299781  PMID: 22428028
12.  Standardizing Data Collection in Traumatic Brain Injury 
Journal of Neurotrauma  2011;28(2):177-187.
Abstract
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.
doi:10.1089/neu.2010.1617
PMCID: PMC3037806  PMID: 21162610
clinical studies; common data elements; data coding; data collection; standardization; traumatic brain injury
13.  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.
Abstract
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.
doi:10.1089/neu.2009.1227
PMCID: PMC2942875  PMID: 20486807
astrocyte; electroconvulsive shock; microglia; seizure; traumatic brain injury
14.  Albumin activates astrocytes and microglia through mitogen-activated protein kinase pathways 
Brain research  2009;1313C:222-231.
Following acute brain injury, albumin may gain access to the brain parenchyma. Clinical studies indicate a protective role for albumin in stroke but an increase in mortality associated with albumin administration following traumatic brain injury. We investigated the effects of albumin on astrocyte and microglial activation, and the role of mitogen activated protein kinases (MAPK) in these responses. Albumin activated ERK1/2, p38 MAPK and JNK signaling pathways in astrocytes, and induced the production of interleukin (IL)-1β, inducible nitric oxide (NO) synthase, the NO metabolite nitrite, and the chemokine CX3CL1 while reducing the level of S100B. The release of inflammatory markers by astrocytes was partially dependent on p38 MAPK and ERK1/2 pathways, but not JNK. In microglia, albumin exposure activated all three MAPK pathways and produced an increase in IL-1 β and nitrite. Inhibition of p38 MAPK in microglia lead to an increased level of IL1β, while inhibition of all three MAPKs suppressed the release of nitrite. These results suggest that albumin activates astrocytes and microglia, inducing inflammatory responses involved both in the mechanisms of cellular injury and repair via activation of MAPK pathways, and thereby implicate glial activation in the clinical responses to administration of albumin.
doi:10.1016/j.brainres.2009.11.063
PMCID: PMC2812578  PMID: 19961838
Albumin; astrocyte; microglia; cytokines; mitogen activated protein kinase
15.  A Rapid Murine Coma and Behavior Scale for Quantitative Assessment of Murine Cerebral Malaria 
PLoS ONE  2010;5(10):e13124.
Background
Cerebral malaria (CM) is a neurological syndrome that includes coma and seizures following malaria parasite infection. The pathophysiology is not fully understood and cannot be accounted for by infection alone: patients still succumb to CM, even if the underlying parasite infection has resolved. To that effect, there is no known adjuvant therapy for CM. Current murine CM (MCM) models do not allow for rapid clinical identification of affected animals following infection. An animal model that more closely mimics the clinical features of human CM would be helpful in elucidating potential mechanisms of disease pathogenesis and evaluating new adjuvant therapies.
Methodology/Principal Findings
A quantitative, rapid murine coma and behavior scale (RMCBS) comprised of 10 parameters was developed to assess MCM manifested in C57BL/6 mice infected with Plasmodium berghei ANKA (PbA). Using this method a single mouse can be completely assessed within 3 minutes. The RMCBS enables the operator to follow the evolution of the clinical syndrome, validated here by correlations with intracerebral hemorrhages. It provides a tool by which subjects can be identified as symptomatic prior to the initiation of trial treatment.
Conclusions/Significance
Since the RMCBS enables an operator to rapidly follow the course of disease, label a subject as affected or not, and correlate the level of illness with neuropathologic injury, it can ultimately be used to guide the initiation of treatment after the onset of cerebral disease (thus emulating the situation in the field). The RMCBS is a tool by which an adjuvant therapy can be objectively assessed.
doi:10.1371/journal.pone.0013124
PMCID: PMC2948515  PMID: 20957049
16.  Enhanced microglial activation and proinflammatory cytokine upregulation are linked to increased susceptibility to seizures and neurologic injury in a ‘two-hit’ seizure model 
Brain research  2009;1282:162-172.
Early-life seizures result in increased susceptibility to seizures and greater neurologic injury with a second insult in adulthood. The mechanisms which link seizures in early-life to increased susceptibility to neurologic injury following a ‘second hit’ are not known. We examined the contribution of microglial activation and increased proinflammatory cytokine production to the subsequent increase in susceptibility to neurologic injury using a kainic acid (KA)-induced, established ‘two-hit’ seizure model in rats. Postnatal day (P)15 rats were administered intraperitoneal KA (early-life seizures) or saline, followed on P45 with either a ‘second hit’ of KA, a first exposure to KA (adult seizures), or saline. We measured the levels of proinflammatory cytokines (IL-1β, TNF-α, and S100B), the chemokine CCL2, microglial activation, seizure susceptibility and neuronal outcomes in adult rats 12 hours and 10 days after the second hit on P45. The ‘two-hit’ group exposed to KA on both P15 and P45 had higher levels of cytokines, greater microglial activation, and increased susceptibility to seizures and neurologic injury compared to the adult seizures group. Treatment after early-life seizures with Minozac, a small molecule experimental therapeutic that targets up-regulated proinflammatory cytokine production, attenuated the enhanced microglial and cytokine responses, the increased susceptibility to seizures, and the greater neuronal injury in the ‘two-hit’ group. These results implicate microglial activation as one mechanism by which early-life seizures contribute to increased vulnerability to neurologic insults in adulthood, and indicate the potential longer term benefits of early-life intervention with therapies that target up-regulation of proinflammatory cytokines.
doi:10.1016/j.brainres.2009.05.073
PMCID: PMC2739829  PMID: 19501063
microglia; cytokines; epilepsy; therapeutic; chemokine
17.  Chronic upregulation of activated microglia immunoreactive for galectin-3/Mac-2 and nerve growth factor following diffuse axonal injury 
Background
Diffuse axonal injury in patients with traumatic brain injury (TBI) can be associated with morbidity ranging from cognitive difficulties to coma. Magnetic resonance imaging scans now allow early detection of axonal injury following TBI, and have linked cognitive disability in these patients to white matter signal changes. However, little is known about the pathophysiology of this white matter injury, and the role of microglial activation in this process. It is increasingly recognized that microglia constitute a heterogeneous population with diverse roles following injury. In the present studies, we tested the hypothesis that following diffuse axonal injury involving the corpus callosum, there is upregulation of a subpopulation of microglia that express the lectin galectin-3/Mac-2 and are involved in myelin phagocytosis.
Methods
Adult mice were subject to midline closed skull injury or sham operation and were sacrificed 1, 8, 14 or 28 days later. Immunohistochemistry and immunofluorescence techniques were used to analyze patterns of labelling within the corpus callosum qualitatively and quantitatively.
Results
Activated microglia immunoreactive for galectin-3/Mac-2 were most abundant 1 day following injury. Their levels were attenuated at later time points after TBI but still were significantly elevated compared to sham animals. Furthermore, the majority of galectin-3/Mac-2+ microglia were immunoreactive for nerve growth factor in both sham and injured animals.
Conclusions
Our results suggest that galectin-3/Mac-2+ microglia play an important role in the pathogenesis of diffuse axonal injury both acutely and chronically and that they mediate their effects, at least in part by releasing nerve growth factor.
doi:10.1186/1742-2094-7-32
PMCID: PMC2891720  PMID: 20507613
18.  Human Herpes Virus 6B: A possible Role in Epilepsy? 
Epilepsia  2008;49(11):1828-1837.
Nearly ubiquitous infection, including central nervous system invasion, with human herpesvirus 6 occurs in childhood. There are two variants, HHV6A and HHV6B. Usually asymptomatic, it is associated with the common, self-limited childhood illness roseola infantum, and rarely with more severe syndromes. In patients with immune compromise, reactivation of viral activity may lead to severe limbic encephalitis. HHV6 has been identified as a possible etiologic agent in multiple sclerosis. A preponderance of evidence supports an association between HHV6 and febrile seizures. An ongoing multicenter study is investigating possible links between HHV6 infection, febrile status epilepticus, and development of mesial temporal sclerosis (MTS). Investigation of temporal lobectomy specimens showed evidence of active HHV6B but not HHV6A replication in hippocampal astrocytes in about two-thirds of patients with MTS, but not other causes of epilepsy. HHV6 has been detected in similar clinical syndromes by other investigators, although less frequently, and additional viruses have been detected as well. It has been suggested that HHV6B may cause ‘excitotoxicity’ by interfering with astrocyte excitatory amino acid transport. Although conventional inflammatory changes are not found in most MTS specimens, inflammatory modulators may play a role in neuronal injury leading to MTS as well. If the link between early viral infection and later development of intractable temporal lobe epilepsy is confirmed, possibly associated with febrile seizures, new therapeutic approaches to a common intractable epilepsy syndrome may be possible.
doi:10.1111/j.1528-1167.2008.01699.x
PMCID: PMC2694582  PMID: 18627418
19.  Pediatric Endocarditis and Stroke: A Single Center Retrospective Review of Seven Cases 
Pediatric neurology  2008;38(4):243-247.
The approach to the acute management of stroke in children with infective endocarditis is limited by the paucity of published data on their clinical course and outcome. We conducted a retrospective study at an urban tertiary care academic center to characterize the clinical course of seven pediatric patients with endocarditis and subsequent cerebral infarct. Among 115 patients with endocarditis, a stroke occurred in seven. Four patients had congenital heart disease. In 6 patients, the stroke was in the distribution of the middle cerebral artery and there was no preference for left or right hemisphere. The most common presenting symptom was focal weakness. Three patients had mycotic aneurysms, all of which were successfully repaired. Two patients were placed on aspirin therapy with no adverse effects. All patients survived but neurological recovery was variable. The two youngest patients (3 and 14 weeks of age) had the longest periods of hospitalization and had the most severe neurologic impairment. These findings suggest that children may have better outcome than adults after stroke secondary to bacterial endocarditis. Routine surveillance for mycotic aneurysms in patients with new neurological deficits and the use of aspirin should be considered in the medical management.
doi:10.1016/j.pediatrneurol.2007.12.009
PMCID: PMC2409276  PMID: 18358401
20.  Suppression of acute proinflammatory cytokine and chemokine upregulation by post-injury administration of a novel small molecule improves long-term neurologic outcome in a mouse model of traumatic brain injury 
Background
Traumatic brain injury (TBI) with its associated morbidity is a major area of unmet medical need that lacks effective therapies. TBI initiates a neuroinflammatory cascade characterized by activation of astrocytes and microglia, and increased production of immune mediators including proinflammatory cytokines and chemokines. This inflammatory response contributes both to the acute pathologic processes following TBI including cerebral edema, in addition to longer-term neuronal damage and cognitive impairment. However, activated glia also play a neuroprotective and reparative role in recovery from injury. Thus, potential therapeutic strategies targeting the neuroinflammatory cascade must use careful dosing considerations, such as amount of drug and timing of administration post injury, in order not to interfere with the reparative contribution of activated glia.
Methods
We tested the hypothesis that attenuation of the acute increase in proinflammatory cytokines and chemokines following TBI would decrease neurologic injury and improve functional neurologic outcome. We used the small molecule experimental therapeutic, Minozac (Mzc), to suppress TBI-induced up-regulation of glial activation and proinflammatory cytokines back towards basal levels. Mzc was administered in a clinically relevant time window post-injury in a murine closed-skull, cortical impact model of TBI. Mzc effects on the acute increase in brain cytokine and chemokine levels were measured as well as the effect on neuronal injury and neurobehavioral function.
Results
Administration of Mzc (5 mg/kg) at 3 h and 9 h post-TBI attenuates the acute increase in proinflammatory cytokine and chemokine levels, reduces astrocyte activation, and the longer term neurologic injury, and neurobehavioral deficits measured by Y maze performance over a 28-day recovery period. Mzc-treated animals also have no significant increase in brain water content (edema), a major cause of the neurologic morbidity associated with TBI.
Conclusion
These results support the hypothesis that proinflammatory cytokines contribute to a glial activation cycle that produces neuronal dysfunction or injury following TBI. The improvement in long-term functional neurologic outcome following suppression of cytokine upregulation in a clinically relevant therapeutic window indicates that selective targeting of neuroinflammation may lead to novel therapies for the major neurologic morbidities resulting from head injury, and indicates the potential of Mzc as a future therapeutic for TBI.
doi:10.1186/1742-2094-5-28
PMCID: PMC2483713  PMID: 18590543
21.  A NITRIC OXIDE DONOR REDUCES BRAIN INJURY AND ENHANCES RECOVERY OF CEREBRAL BLOOD FLOW AFTER HYPOXIA-ISCHEMIA IN THE NEWBORN RAT 
Neuroscience letters  2007;415(2):124-129.
Nitric oxide (NO) released in response to hypoxia-ischemia (HI) in the newborn brain may mediate both protective and pathologic responses. We sought to determine whether pharmacologic increase of NO using an NO donor would reduce neurologic injury resulting from HI in the postnatal day 7 rat. We measured NO levels and CBF in the presence of either a NOS inhibitor, N-nitro-L-arginine methyl ester (L-NAME) or an NO donor (Z)-1-[N-(2aminoethyl)-N-(2-ammonio-ethyl)amino]diazen-1-ium-1,2-diolate (DETANONOate). Both inhibition of NOS and administration of an NO donor reduced neuropathologic injury after 7-day recovery. NO levels decreased in both ischemic and contralateral hemispheres during HI. This response was prevented by treatment with DETANONOate. Despite the decrease in NO, CBF increased during ischemia in the contralateral hemisphere but decreased when combined with brief hypoxia. Treatment with L-NAME abolished these increases, which were not altered by DETANONOate. Reduction of cellular metabolism by mild hypothermia also reduced both NO and CBF. Following prolonged HI, CBF remained decreased in the ischemic hemisphere up to 24-hour recovery. This decrease was prevented by treatment with DETANONOate. These data show that administration of an NO donor reduces neurologic injury following HI in the newborn rat. This mechanism of this protection, in part, is due to an increase in the rate of recovery of CBF compared to vehicle-treated animals. Augmentation of NO-dependent increases in CBF may serve to improve neurologic outcome after perinatal asphyxia.
doi:10.1016/j.neulet.2007.01.019
PMCID: PMC1857363  PMID: 17270345
Hypoxia-ischemia; nitric oxide; cerebral blood flow; hypothermia; newborn
22.  Uptake and cell-killing activities of a series of Victoria blue derivatives in a mouse mammary tumour cell line 
Cytotechnology  1999;29(1):35-43.
The triarylmethane dye Victoria blue BO (VBBO) is a known photosensitizer which has been shown to induce a cytotoxic response in vitro. Several novel Victoria blue derivatives, with varying physicochemical properties, have been compared to VBBO, with respect both to dark toxicity and phototoxicity, on a mouse mammary tumour cell line, EMT6. Photosensitizer uptake was observed using confocal fluorescence microscopy. The chemical differences, particularly in the naphthyl substitution of the derivatives were shown to alter the light:dark toxicity differential and the uptake of the photosensitizers.
doi:10.1023/A:1008098810928
PMCID: PMC3449467  PMID: 19003335
confocal microscopy; mammary tumour; photosensitizers; Victoria bleu

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