Search

The use of mass spectrometry for protein identification and quantification in cerebrospinal fluid (CSF) is at the forefront of research efforts to identify and explore biomarkers for the early diagnosis and prognosis of neurologic disorders. Here we implemented a 4-plex N,N-dimethyl leucine (DiLeu) isobaric labeling strategy in a longitudinal study aiming to investigate protein dynamics in children with B-cell acute lymphoblastic leukemia (B-cell ALL) undergoing chemotherapy. The temporal profile of CSF proteome during chemotherapy treatment at weeks 5, 10-14, and 24-28 highlighted many differentially expressed proteins, such as neural cell adhesion molecule, neuronal growth regulator 1, and secretogranin-3, all of which play important roles in neurodegenerative diseases. A total of 63 proteins were significantly altered across all of the time points investigated. The most over-represented biological processes from gene ontology analysis included platelet degranulation, complement activation, cell adhesion, fibrinolysis, neuron projection, regeneration, and regulation of neuron death. We expect that results from this and future studies will provide a means to monitor neurotoxicity and develop strategies to prevent central nervous system injury in response to chemotherapy in children.

The International League Against Epilepsy/American Epilepsy Society (ILAE/AES) Joint Translational Task Force initiated the TASK3 working group to create common data elements (CDEs) for various aspects of preclinical epilepsy research studies, which could help improve the standardization of experimental designs. This article addresses neuropathological changes associated with seizures and epilepsy in rodent models of epilepsy. We discuss CDEs for histopathological parameters for neurodegeneration, changes in astrocyte morphology and function, mechanisms of inflammation, and changes in the blood-brain barrier and myelin/oligodendrocytes resulting from recurrent seizures in rats and mice. We provide detailed CDE tables and case report forms (CRFs), and with this companion manuscript, we discuss the rationale and methodological aspects of individual neuropathological examinations. The CDEs, CRFs, and companion paper are available to all researchers, and their use will benefit the harmonization and comparability of translational preclinical epilepsy research. The ultimate hope is to facilitate the development of rational therapy concepts for treating epilepsies, seizures, and comorbidities and the development of biomarkers assessing the pathological state of the disease.

Simple Summary In childhood leukemias the central nervous system (CNS) can be invaded by leukemic cells, leading to disease recurrence and treatment failures. Analysis of the cerebrospinal fluid (CSF), which surrounds the brain and spinal cord, is crucial for detection of leukemia in that compartment. Despite advances made in treatments for childhood leukemias, therapies continue to adversely affect the developing brain with resulting neurocognitive deficits in many survivors. This review presents an overview of studies demonstrating that CSF proteins, nucleic acids and metabolites, so called biomarkers, can be utilized in tracing both CNS disease and neurotoxicity of treatments in childhood leukemias. Abstract Involvement of the central nervous system (CNS) in childhood leukemias remains a major cause of treatment failures. Analysis of the cerebrospinal fluid constitutes the most important diagnostic pillar in the detection of CNS leukemia and relies primarily on cytological and flow-cytometry studies. With increasing survival rates, it has become clear that treatments for pediatric leukemias pose a toll on the developing brain, as they may cause acute toxicities and persistent neurocognitive deficits. Preclinical research has demonstrated that established and newer therapies can injure and even destroy neuronal and glial cells in the brain. Both passive and active cell death forms can result from DNA damage, oxidative stress, cytokine release, and acceleration of cell aging. In addition, chemotherapy agents may impair neurogenesis as well as the function, formation, and plasticity of synapses. Clinical studies show that neurocognitive toxicity of chemotherapy is greatest in younger children. This raises concerns that, in addition to injury, chemotherapy may also disrupt crucial developmental events resulting in impairment of the formation and efficiency of neuronal networks. This review presents an overview of studies demonstrating that cerebrospinal fluid biomarkers can be utilized in tracing both CNS disease and neurotoxicity of administered treatments in childhood leukemias.

Studies in animal models have demonstrated that long exposures to anesthetics can induce apoptosis in the newborn and young developing brain. These effects have not been confirmed in humans due to the lack of a non-invasive, practical in vivo imaging tool with the ability to detect these changes. Following the success of using Ultrasound Backscatter Spectroscopy (UBS) to monitor in vivo cell death in breast tumors, we aimed at applying UBS to assess neurotoxicity of the anesthetic Sevoflurane (SEVO) in a non-human primate (NHP) model. Sixteen 2–7 day-old Rhesus macaques were exposed for 5 hours to SEVO. Ultrasound scanning was done with a phased array transducer on a clinical ultrasound scanner operated at 10 MHz. Data consisting of 10–15 frames of radiofrequency (RF) echo signals from coronal views of the thalamus were obtained at 0.5 and 6.0 h after initiating exposure. The UBS parameter “Effective Scatterer Size” (ESS) was estimated by fitting a scattering form factor (FF) model to the FF measured from RF echo signals. The approach involved analyzing the frequency-dependence of the measured FF to characterize scattering sources and selecting the FF model based on a χ(2) goodness of fit criterion. To assess data quality, a rigorous acceptance criterion based on the analysis of prevalence of diffuse scattering (an assumption in the estimation of ESS) was established. ESS changes after exposure to SEVO were compared to changes in a control group of 5 primates for which ultrasound data were acquired at 0 min and 10 min (no apoptosis expected). Over the entire data set, the average measured FF at 0.5 h and 6.0 h monotonically decreased with frequency, justifying fitting a single form factor over the analysis bandwidth. χ(2) values of an (inhomogeneous continuum) Gaussian FF model were one fifth those of the discrete fluid sphere model, suggesting that a continuum scatterer model better represents ultrasound scattering in the young Rhesus brain. After applying the data quality criterion, only 5 out of 16 subjects from the apoptotic group, and 5 out of 5 subjects from the control group, fulfilled the acceptance criteria. All of the subjects in the apoptotic group that passed the acceptance criterion showed a significant ESS reduction at 6.0 h. These changes (−6.4% [95% Confidence Interval −14.3% to −3.3%]) were larger than those in the control group (−0.8% [95% Confidence Interval −2.0% to 1.5%]). Data with low prevalence of diffuse scattering corresponded to possibly biased results. Thus, ESS has potential to detect changes in brain microstructure related to anesthesia-induced apoptosis.

Background: Glutamate receptors are widely expressed in different types of cancer cells. α-Amino-3- hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors are ionotropic glutamate receptors which are coupled to intracellular signaling pathways that influence cancer cell survival, proliferation, and migration. Blockade of AMPA receptors by pharmacologic compounds may potentially constitute an effective tool in anticancer treatment strategies. Method: Here we investigated the impact of the AMPA receptor antagonist CFM-2 on the expression of the protein survivin, which is known to promote cancer cell survival and proliferation. We show that CFM-2 inhibits survivin expression at mRNA and protein levels and decreases the viability of cancer cells. Using a stably transfected cell line which overexpresses survivin, we demonstrate that over-expression of survivin enhances cancer cell viability and attenuates CFM-2–mediated inhibition of cancer cell growth. Result: These findings point towards suppression of survivin expression as a new mechanism contributing to anticancer effects of AMPA antagonists.

A male neonate is born via vaginal delivery at 37 1/7 weeks’ gestation to a healthy 30-year-old gravida 3, para 2 mother. The pregnancy had been complicated by polyhydramnios, severe fetal hydronephrosis, anti-Kell antibodies, and questionable fetal mega cisterna magna. Labor is induced secondary to worsening bilateral fetal hydronephrosis. The delivery is uncomplicated and Apgar scores are 9 and 9 at 1 and 5 minutes, respectively. Initial physical examination findings are significant only for left-sided club foot, and the infant is admitted to the well-newborn nursery. State newborn screening is performed 24 hours after birth, results of which are normal. There is no significant familial history of congenital neurologic or urologic abnormalities, and both previous children of these parents are healthy. Amniocentesis performed during this pregnancy secondary to the bilateral fetal hydronephrosis had revealed a normal fetal microarray. The infant is admitted to the NICU 3 days after birth secondary to hypernatremia (150 mEq/L [150 mmol/L]) and severe (grade 4) bilateral hydronephrosis confirmed with postnatal ultrasonography. While in the NICU, the neonate is noted to have episodes of lateral eye deviation, bilateral arm and leg twitching, and episodes of apnea. Electrolytes (excluding hypernatremia), glucose, and cranial ultrasonography findings are normal. A sepsis evaluation is completed and the infant is treated for 48 hours with ampicillin, cefotaxime, and acyclovir until blood, urine, and cerebrospinal fluid cultures return negative. Amplitude-integrated electroencephalography (EEG) findings are concerning for seizure activity and video EEG shows frequent multifocal epileptiform activity in both hemispheres indicative of abnormal cortical hyperexcitability, lowering the threshold for seizures. Following consultation with pediatric neurology he receives a loading dose of levetiracetam and maintenance therapy is initiated. Brain magnetic resonance imaging (MRI) reveals trace right occipital subarachnoid hemorrhage and trace infratentorial posterior fossa subdural hematoma thought to be secondary to birth trauma and …

Spinal cord injury (SCI) and the consecutive devastating neurological sequelae have an enormous individual and economic impact. Implantation of functionalized hydrogels is a promising approach, because they can serve as a matrix for the regenerating tissue, carry and release bioactive molecules and various cell types. We already demonstrated that non-functionalized soft alginate hydrogel supported axonal outgrowth and protected neurons against oxidative stress in vitro. Here, we investigated the effects of such soft alginate hydrogels on locomotor recovery in small and large spinal cord lesions. Hemimyelonectomy of 2 mm or 4 mm length was performed in rats and soft alginate hydrogel was implanted. Functional recovery of the hindlimbs was assessed in the open field [Batto Beattie Bresnahan (BBB) score] and using swimming test [Louisville Swim score (LSS)] for 140 days post injury (DPI). Reference histology was performed. Rats that received an alginate implant into 2 mm spinal cord lesions demonstrated significantly improved locomotor recovery compared to controls detectable already at 10 DPI. At 140 DPI, they reached higher LSS and BBB scores in swimming and open field tests, respectively. However, this beneficial effect of alginate was lacking in animals with larger (4 mm) lesions. Histological examination suggested that fibrous scarring in the spinal cord was reduced after alginate implantation in comparison to controls. Implantation of soft alginate hydrogel in small spinal cord lesions improved functional recovery. Possible underlying mechanisms include the mechanical stabilization of the wound, reduction of secondary damage and inhibition of fibrous scarring.

This talk will present recent results from our multi-institutional effort to advance the application of Quantitative Ultrasound in obstetrics and in perinatal care. In the field of obstetrics, we are investigating the use of shear wave elasticity imaging (SWEI) to predict the risk of preterm birth based on assessing the viscoelastic properties of the uterine cervix. Longitudinal studies in non-human primates (NHP) and humans showed a similar rate of decrease of the shear wave speed, SWS (1.6% and 1.5%/% gestational age, respectively), validating the animal model and indicating the value of SWS change as a biomarker for cervical softening. We are currently optimizing the analysis of shear wave dispersion to assess changes in viscosity. In the field of perinatal care, results from a pilot study in NHP neonates suggest that the effective size of diffuse scatterers can be used as a biomarker for thalamic apoptosis induced by long exposures to anesthesia. We are currently investigating the use of coherent scattering features to increase the sensitivity and specificity to neuroapoptosis and to improve attenuation and backscatter coefficient estimations in complex tissues such as the cervix and the neonate brain. [Acknowledgments: All protocols have been IRB- and/or IACUC-approved, and are HIPAA compliant. Equipment loan and technical support from Siemens Healthcare. Funded by: NIH Grant Nos. T32CA009206, F31HD082911, R01HD072077, UL1TR000427, P51OD011106, and R01HD083001; NSERC Grant No. RGPIN-2015-04136; and UNAM-PAPIIT IA104518 and IN107916.This talk will present recent results from our multi-institutional effort to advance the application of Quantitative Ultrasound in obstetrics and in perinatal care. In the field of obstetrics, we are investigating the use of shear wave elasticity imaging (SWEI) to predict the risk of preterm birth based on assessing the viscoelastic properties of the uterine cervix. Longitudinal studies in non-human primates (NHP) and humans showed a similar rate of decrease of the shear wave speed, SWS (1.6% and 1.5%/% gestational age, respectively), validating the animal model and indicating the value of SWS change as a biomarker for cervical softening. We are currently optimizing the analysis of shear wave dispersion to assess changes in viscosity. In the field of perinatal care, results from a pilot study in NHP neonates suggest that the effective size of diffuse scatterers can be used as a biomarker for thalamic apoptosis induced by long exposures to anesthesia. We are currently investigating the use of coherent scat...

Background In utero Zika virus (ZIKV) infection causes birth defects and neurodevelopmental deficits in neonates. We reasoned that a translational macaque model of congenital ZIKV infection could define disease pathophysiology not possible in human clinical studies. Methods We inoculated 5 pregnant rhesus macaques with a Puerto Rican isolate of ZIKV (ZIKV-PRVABC59) during the first trimester, monitored infection with plasma viral RNA (vRNA) loads, and evaluated infants for birth defects and neurodevelopmental deficits during their first week of life. Assessments included neurobehavioral assessments, ophthalmic examinations, optical coherence tomography, electroretinography with visual evoked potentials, hearing examinations, brain magnetic resonance imaging (MRI), and tissue histopathological analyses. Results All five pregnant dams demonstrated plasma viremia and seroconversion following ZIKV inoculation. One of the five pregnancies resulted in a stillbirth. All liveborn infants had decreased feeding volumes and weight gain compared with control infants. A comprehensive voxel-based morphometric comparison of ZIKV-exposed and control infant brain MRIs identified increased gray matter volume and density in the frontal lobe in the ZIKV-exposed infant group, which corresponds to the pharyngeal motor cortex responsible for coordinating swallowing. Ocular studies identified differences between ZIKV-exposed and control infants in retinal layer thicknesses (inner plexiform, outer nuclear layers, photoreceptor outer segment) and visual evoked potentials (increased amplitude of waveforms). While ZIKV vRNA was detected in the decidua of 2/5 pregnancies, no ZIKV vRNA was identified in infant tissues and none of the infants developed an anti-ZIKV IgM response. Conclusion In utero ZIKV exposure resulted in decreased feeding volumes and weight gain, which may be related to the gray matter changes identified in the pharyngeal motor cortex. Changes in retinal layer thicknesses and increased cortical visual pathway waveform amplitude suggest vision may be impaired. These changes occurred despite the lack of evidence of vertical transmission, suggesting that ZIKV exposure without measurable vertical transmission affects fetal brain development. Disclosures All authors: No reported disclosures.

Objective: Though central nervous system irritability is a well-established consequence of neonatal drug withdrawal, brain function in infants with neonatal abstinence syndrome (NAS) is not well un...

Sedatives and anesthetics can injure the developing brain. They cause apoptosis of neurons and oligodendrocytes, impair synaptic plasticity, inhibit neurogenesis and trigger long-term neurocognitive deficits. The projected vulnerable period in humans extends from the third trimester of pregnancy to the third year of life. Despite all concerns, there is no ethically and medically acceptable alternative to the use of sedatives and anesthetics for surgeries and painful interventions. Development of measures that prevent injury while allowing the medications to exert their desired actions has enormous translational value. Here we investigated protective potential of hypothermia against histological toxicity of the anesthetic sevoflurane in the developing nonhuman primate brain. Neonatal rhesus monkeys underwent sevoflurane anesthesia over 5hrs. Body temperature was regulated in the normothermic (>36.5°C), mild hypothermic (35–36.5°C) and moderately hypothermic (

Glycans are highly complex entities with multiple building units and different degrees of branched polymerization. Intensive research efforts have been directed to mass spectrometry (MS)-based qualitative and quantitative glycomic analysis due to the important functions of glycans. Among various strategies, isobaric labeling has become popular because of its higher multiplexing capacity. Over the past few years, several isobaric chemical tags have been developed for quantitative glycomics. However, caveats also exist for these tags, such as relatively low reporter ion yield for aminoxyTMT-labeled complex glycans. To overcome the limitations of existing isobaric chemical tags, we designed a class of novel isobaric multiplex reagents for carbonyl-containing compound (SUGAR) tags that can be used to label glycans for quantitative glycomic analysis. The quantitative performance including labeling efficiency, quantification accuracy, and dynamic range of these SUGAR tags has been evaluated, showing promising results. Finally, the 4-plex SUGAR tags have been utilized to investigate N-glycan changes of B-cell acute lymphoblastic leukemia (ALL) pediatric patients before and after chemotherapy.

Neurologic dysfunction during acute lymphoblastic leukemia treatment is commonly associated with chemotherapy. Nonchemotherapy contributions should be considered for persistent atypical symptoms. We describe a boy with acute lymphoblastic leukemia who developed recurrent fevers, diarrhea, progressive ataxia, and neuropsychiatric impairment during maintenance chemotherapy. He was found to have cytomegalovirus in his serum and colon, but not in his cerebrospinal fluid. Instead, his cerebrospinal fluid revealed oligoclonal bands not present in the serum, suggesting an autoimmune process. Prompt treatment with ganciclovir and immunotherapy resulted in marked clinical improvement. Early recognition and treatment of an autoimmune encephalitis is paramount for optimal clinical outcome.

Survival rates of children with cancer are steadily increasing. This urges our attention to neurocognitive and psychiatric outcomes, as these can markedly influence the quality of life of these children. Neurobehavioral morbidity in childhood cancer survivors affects diverse aspects of cognitive function, which can include attention, memory, processing speed, intellect, academic achievement, and emotional health. Reasons for neurobehavioral morbidity are multiple with one major contributor being chemotherapy-induced central nervous system (CNS) toxicity. Clinical studies investigating the effects of chemotherapy on the CNS in children with cancer have reported causative associations with the development of leukoencephalopathies as well as smaller regional grey and white matter volumes, which have been found to correlate with neurocognitive deficits. Preclinical work has provided compelling evidence that chemotherapy drugs are potent neuro- and gliotoxins in vitro and in vivo and can cause brain injury via excitotoxic and apoptotic mechanisms. Furthermore, chemotherapy triggers DNA (deoxyribonucleic acid) damage directly or through increased oxidative stress. It can shorten telomeres and accelerate cell aging, cause cytokine deregulation, inhibit hippocampal neurogenesis, and reduce brain vascularization and blood flow. These mechanisms, when allowed to operate on the developing brain of a child, have high potential to not only cause brain injury, but also alter crucial developmental events, such as myelination, synaptogenesis, neurogenesis, cortical thinning, and formation of neuronal networks. This short review summarizes key publications describing neurotoxicity of chemotherapy in pediatric cancers and potential underlying pathomechanisms.

We report the characterization of the thalamus of fourteen newborn Rhesus macaques using incoherent and coherent ultrasound scattering. Particularly, we study whether sources of coherent scattering could contribute to variability of estimates of the effective scatterer size, ESS, used to detect neurotoxic effects of anesthesia. This characterization is performed by computing the generalized spectrum, the echo-amplitude signal-to-noise ratio, and the echo phase histogram nonuniformity, and comparing their values to those obtained from a reference material with diffuse scattering conditions. Results show that scattering conditions in the thalamus can be described mostly as originating from a low scatterer number density (38% of data) and the presence of specular reflectors (36%). Only 23 % of the data presented diffuse scattering conditions. Specular reflectors were more likely found in data that showed inconclusive results in a previous ESS analysis of the effects of anesthesia. Future work will be focused on implementing coherent scattering descriptors to characterize the thalamus, as well as developing strategies to reduce the effects of this scattering scenario in the estimation of the ESS.

Barbiturates and benzodiazepines are potent GABAA receptor agonists and strong anticonvulsants. In the developing brain they can cause neuronal and oligodendroglia apoptosis, impair synaptogenesis, inhibit neurogenesis and trigger long-term neurocognitive sequelae. In humans, the vulnerable period is projected to extend from the third trimester of pregnancy to the third year of life. Infants with seizures and epilepsies may receive barbiturates, benzodiazepines and their combinations for days, months or years. How exposure duration affects neuropathological sequelae is unknown. Here we investigated toxicity of phenobarbital/midazolam (Pb/M) combination in the developing nonhuman primate brain. Neonatal rhesus monkeys received phenobarbital intravenously, followed by infusion of midazolam over 5 (n = 4) or 24 h (n = 4). Animals were euthanized at 8 or 36 h and brains examined immunohistochemically and stereologically. Treatment was well tolerated, physiological parameters remained at optimal levels. Compared to naive controls, Pb/M exposed brains displayed widespread apoptosis affecting neurons and oligodendrocytes. Pattern and severity of cell death differed depending on treatment-duration, with more extensive neurodegeneration following longer exposure. At 36 h, areas of the brain not affected at 8 h displayed neuronal apoptosis, while oligodendroglia death was most prominent at 8 h. A notable feature at 36 h was degeneration of neuronal tracts and trans-neuronal death of neurons, presumably following their disconnection from degenerated presynaptic partners. These findings demonstrate that brain toxicity of Pb/M in the neonatal primate brain becomes more severe with longer exposures and expands trans-synaptically. Impact of these sequelae on neurocognitive outcomes and the brain connectome will need to be explored.

We recently developed a novel amine-reactive mass-defect-based chemical tag, dimethyl pyrimidinyl ornithine (DiPyrO), for quantitative proteomic analysis at the MS(1) level. In this work, we further extend the application of the DiPyrO tag, which provides amine group reactivity, optical detection capability, and improved electrospray sensitivity, to quantify N-linked glycans enzymatically released from glycoproteins in the glycosylamine form. Duplex DiPyrO tags that differ in mass by 45.3 mDa were used to label the glycosylamine moieties of freshly released N-glycosylamines from glycoprotein standards and human serum proteins. We demonstrate that both MALDI-LTQ-Orbitrap and nano-HILIC LC/MS/MS Fusion Lumos Orbitrap platforms are capable of resolving the singly or multiply charged N-glycans labeled with mass-defect DiPyrO tags. Dynamic range of quantification, based on MS(1) peak intensities, was evaluated across 2 orders of magnitude. With optimized N-glycan release conditions, glycosylamine labeling conditions, and MS acquisition parameters, the N-glycan profiles and abundances in human serum proteins of cancer patients before and after chemotherapy were compared. Moreover, this study also opens a door for using well-developed amine-reactive tags for relative quantification of glycans, which could be widely applied.

Caffeine is the most frequently used medication in premature infants. It is the respiratory stimulant of choice for apnea associated with prematurity and has been called the silver bullet in neonatology because of many proven benefits and few known risks. Research has revealed that sedative/anesthetic drugs trigger apoptotic death of neurons and oligodendrocytes in developing mammalian brains. Here we evaluated the influence of caffeine on the neurotoxicity of anesthesia in developing nonhuman primate brains. Fetal macaques (n = 7–8/group), at a neurodevelopmental age comparable to premature human infants, were exposed in utero for 5 hours to no drug (control), isoflurane, or isoflurane + caffeine and examined for evidence of apoptosis. Isoflurane exposure increased apoptosis 3.3 fold for neurons and 3.4 fold for oligodendrocytes compared to control brains. Isoflurane + caffeine caused neuronal apoptosis to increase 8.0 fold compared to control levels but did not augment oligoapoptosis. Neuronal death was particularly pronounced in the basal ganglia and cerebellum. Higher blood levels of caffeine within the range considered therapeutic and safe for human infants correlated with increased neuroapoptosis. Caffeine markedly augments neurotoxicity of isoflurane in the fetal macaque brain and challenges the assumption that caffeine is safe for premature infants.

BACKGROUND High definition transcranial direct current stimulation (HD-tDCS) has been administered over single brain regions for small numbers of sessions. Safety, feasibility and tolerability of HD-tDCS over multiple brain regions, multiple daily stimulations and long periods are not established. OBJECTIVE We studied safety, feasibility and tolerability of daily HD-tDCS over 2-4 brain regions for 20 sessions in healthy adults. METHODS Five healthy adults underwent physical and neurological examination, electrocardiogram (EKG), electroencephalogram (EEG) and cognitive screening (ImpACT) before, during and after HD-tDCS. Four networks (left/right temporoparietal and frontal) were stimulated in sequence (20 min each) using HD-tDCS in 20 daily sessions. Sessions 1-10 included sequential stimulation of both temporoparietal networks, sessions 11-15 stimulations of 4 networks and sessions 16-20 two daily stimulation cycles of 4 networks/cycle (1.5 mA/network). Side effects, ImpACT scores and EEG power spectrum were compared before and after HD-tDCS. RESULTS All subjects completed the trial. Adverse events were tingling, transient redness at the stimulation site, perception of continuing stimulation after end of session and one self-resolving headache. EEG power spectrum showed decreased delta power in frontal areas several days after HD-tDCS. While at the group level ImpACT scores did not differ before and after stimulations, we found a trend for correlation between decreased EEG delta power and individual improvements in ImpACT scores after HD-tDCS. CONCLUSION Prolonged, repeat daily stimulation of multiple brain regions using HD-tDCS is feasible and safe in healthy adults. Preliminary EEG results suggest that HD-tDCS may induce long lasting changes in excitability in the brain.

BACKGROUND: Pelizaeus Merzbacher disease (PMD) is a dysmyelinating disorder of the central nervous system caused by impaired differentiation of oligodendrocytes. This study was prompted by findings that antimuscarinic compounds enhance oligodendrocyte differentiation and remyelination in vitro. One of these compounds, clemastine fumarate, is licensed for treatment of allergic conditions. We tested whether clemastine fumarate can promote myelination in two rodent PMD models, the myelin-deficient and the PLP transgenic rat. METHODS: Pups were treated with daily injections of clemastine (10–30 mg/kg/day) on postnatal days 1–21. Neurologic phenotypes and myelination patterns in the brain, optic nerves, and spinal cords were assessed using histological techniques. RESULTS: No changes in neurological phenotype or survival were observed even at the highest dose of clemastine. Postmortem staining with Luxol fast blue and myelin basic protein immunohistochemistry revealed no evidence for improved myelination in the CNS of treated rats compared to vehicle-treated littermates. Populations of mature oligodendrocytes were unaffected by the treatment. CONCLUSION: These results demonstrate lack of therapeutic effect of clemastine in two rat PMD models. Both models have rapid disease progression consistent with the connatal form of the disease. Further studies are necessary to determine whether clemastine bears a therapeutic potential in milder forms of PMD.

Background: Regardless of contemporary improvements in cancer treatment, the results of drug treatment are not always efficacious. Thus, the development of novel approaches that affect cancer cell-specific metabolic pathways is needed. Since much evidence has shown that tumor cell proliferation and motility are stimulated by glutamate via activation of its receptors, use of antagonists to these receptors may be the key to control cancer cell progression. Riluzole noncompetitive metabotropic glutamate receptor 1 (mGluR1) antagonist, commonly used to treat patients with amyotrophic lateral sclerosis (ALS), has shown some antineoplastic properties against melanoma, breast and prostate cancer. Yet little is known about its molecular mode of action. Aims: The current study aims at evaluating the abilities of Riluzole to inhibit proliferation of several cancer cell lines, as well as resolve the mechanism of its action. Method: We demonstrated antiproliferative and antimigrative properties of Riluzole in rhabdomyosarcomamedulloblastoma, neuroblastoma, astrocytoma, glioma, colon cancer, lung cancer, thyroid carcinoma, leukemia, erythroleukemia and multiple myeloma. Our studies revealed apoptosis induction and G2-M cell cycle arrest in Riluzole treated A549, C6 and HT-29 cells. Result: At the molecular level, we found that these cells treated with Riluzole had a decrease of Cyclin B and an increase of p21 Waf1/Cip1 and p53 expression. We also observed an enhancement of CDK1 and Chk2 phosphorylation. Reported changes may suggest the involvement of these proteins in G2-M arrest, observed in flow cytometry analysis. These data indicated the potential use of Riluzole in the treatment of different types of cancers.

Soft alginate hydrogels support robust neurite outgrowth, but their rapid disintegration in solutions of high ionic strength restricts them from long-term in vivo applications. Aiming to enhance the mechanical stability of soft alginate hydrogels, we investigated how changes in pH and ionic strength during gelation influence the swelling, stiffness, and disintegration of a three-dimensional (3D) alginate matrix and its ability to support neurite outgrowth. Hydrogels were generated from dry alginate layers through ionic crosslinks with Ca(2+) (≤ 10 mM) in solutions of low or high ionic strength and at pH 5.5 or 7.4. High- and low-viscosity alginates with different molecular compositions demonstrated pH and ionic strength-independent increases in hydrogel volume with decreases in Ca(2+) concentrations from 10 to 2 mM. Only soft hydrogels that were synthesized in the presence of 150 mM of NaCl (Ca-alginate NaCl) displayed long-term volume stability in buffered physiological saline, whereas analogous hydrogels generated in NaCl-free conditions (Ca-alginate) collapsed. The stiffnesses of Ca-alginate NaCl hydrogels elevated from 0.01 to 19 kPa as the Ca(2+)-concentration was raised from 2 to 10 mM; however, only Ca-alginate NaCl hydrogels with an elastic modulus ≤ 1.5 kPa that were generated with ≤ 4 mM of Ca(2+) supported robust neurite outgrowth in primary neuronal cultures. In conclusion, soft Ca-alginate NaCl hydrogels combine mechanical stability in solutions of high ionic strength with the ability to support neural growth and could be useful as 3D implants for neural regeneration in vivo.

Glutamate, a nonessential amino acid, is a major bioenergetic substrate for proliferating normal and neoplastic cells on one hand and an excitatory neurotransmitter that is actively involved in biosynthetic, bioenergetic, metabolic, and oncogenic signaling pathways on the other. It exerts its action through a family of receptors consisting of metabotropic glutamate receptors (mGluRs) and ionotropic glutamate receptors (iGluRs), both of which have been implicated previously in a broad spectrum of acute and chronic neurodegenerative diseases. In this review, we discuss existing data on the role of glutamate as a growth factor for neoplastic cells, the expression of glutamate receptors in various types of benign and malignant neoplasms, and the potential roles that GluRs play in cancer development and progression along with their clinical significance. We conclude that glutamate-related receptors and their signaling pathways may provide novel therapeutic opportunities for a variety of malignant human diseases.

Matrix metalloproteinases (MMPs) are involved in tissue repair, cell death and morphogenesis. We investigated the role of the gelatinases MMP-2 and MMP-9 in the pathogenesis of neuronal death induced by prolonged seizures in the developing brain. Seven-day-old rats, MMP-9 knockout mice and transgenic rats overexpressing MMP-9 received intraperitoneal injections of pilocarpine, 250 mg/kg, to induce seizures. After 6-72 h pups were sacrificed, tissue from different brain regions was isolated and expression of MMP-9 mRNA and protein was analyzed by real-time PCR or Western blot. Additionally, brains were fixed and processed for TUNEL-staining, immunohistochemistry and in situ zymography. We found increased numbers of TUNEL-positive cells 24 h after pilocarpine-induced seizures, most pronounced in cortical areas and the dentate gyrus, and less pronounced in thalamus. At 6-24 h, MMP-9 mRNA levels showed significant elevation compared to sham-treated controls; this effect resolved by 48 h, whereas MMP-2 mRNA levels remained stable. Cortical gelatinolytic activity, monitored by in situ zymography, was enhanced following pilocarpine-induced seizures. The MMP inhibitor GM 6001 ameliorated cell death following pilocarpine-induced seizures in infant rats. MMP-9 knockout mice were less susceptible to seizure-induced brain injury. Transgenic rats overexpressing MMP-9 were equally susceptible to seizure-induced brain injury as wild type rats. Our results suggest a significant contribution of MMP-9 to cell death after pilocarpine-induced seizures in the developing brain. As indicated by Western blot analysis, MMP-9 activation may be linked to activation of the Erk/CREB-pathway. The findings implicate involvement of MMP-9 in the pathophysiology of brain injury following seizures in the developing brain.

Cell death occurs physiologically in the mammalian brain during the period of the growth spurt. In humans, this period starts in the 3rd trimester of gestation and ends by the third year of life. Environmental factors can interact with programmed cell death mechanisms to pathologically increase the numbers of neurons undergoing self elimination (apoptosis) and potentially lead to brain injury. It has been shown that classes of drugs which block glutamate N-methyl-D-aspartate (NMDA) receptors, activate γ- aminobutyric-acid (GABA A ) receptors or block voltage gated sodium channels, when administered to immature rodents during susceptible developmental periods, trigger profound apoptotic cell death in the brain. Sedative, anesthetic and anticonvulsant drugs utilize these mechanisms to exert their actions. In addition, short exposures to non-physiologic oxygen levels can also trigger apoptotic cell death in the brains of infant rodents. Pathomechanisms involved in the neurotoxic actions of sedatives, anesthetics, anticonvulsants and oxygen include decreased expression of neurotrophins, inactivation of survival signaling proteins, activation of inflammatory cytokines as well as oxidative stress. These findings raise concerns regarding treatment of pregnant women, infants and young children with sedatives, anesthetics and anticonvulsants and premature infants with oxygen. Modified approaches should be developed for patients within these vulnerable age groups.

Malignant glioma represents the most common primary adult brain tumor in Western industrialized countries. Despite aggressive treatment modalities, the median survival duration for patients with glioblastoma multiforme (GBM), the highest grade malignant glioma, has not improved significantly over past decades. One promising approach to deal with GBM is the inactivation of proteins essential for survival or progression of glioma cells by means of RNA interference (RNAi) techniques. A likely candidate for an RNAi therapy of gliomas is the inhibitor of apoptosis protein survivin. Survivin is involved in 2 main cellular processes-cell division and inhibition of apoptosis. We show here that stable RNAi of survivin induced polyploidy, apoptosis, and impaired proliferation of human U343-MG, U373-MG, H4, and U87-MG cells and of primary glioblastoma cells. Proteome profiler arrays using U373-MG cells identified a novel set of differentially expressed genes upon RNAi-mediated survivin knockdown. In particular, the death receptor TRAIL R2/DR5 was strongly upregulated in survivin-depleted glioma cells, inducing an enhanced cytotoxic response of allogeneic human NK cells. Moreover, an experimental in vivo therapy using polyethylenimine (PEI)/siRNA complexes for survivin knockdown efficiently blocked tumor growth of established subcutaneous U373-MG tumors and enhanced survival of NMRI(nu/nu) mice orthopically transplanted with U87-MG cells. We conclude that survivin is functionally relevant in gliomas and that PEI-mediated exogenous delivery of siRNA targeting survivin is a promising strategy for glioblastoma therapy.

Neonatal seizures are common, especially in prematurity. Phenobarbital (PB) currently represents the antiepileptic drug (AED) of choice, despite being related to increased neuronal apoptosis in animal models and cognitive impairment in human subjects. Levetiracetam (LEV) may have a more favorable profile since it does not cause neuronal apoptosis in infant rodents.In a prospective feasibility study, LEV was applied as first-line treatment in 38 newborns with EEG-confirmed seizures, after ruling out hypoglycemia, hypocalcaemia, hypomagnesaemia and pyridoxin dependency. Initial intravenous doses of 10 mg/kg LEV were gradually increased to 30 mg/kg over 3 days with a further titration to 45-60 mg/kg at the end of the week. Acute intervention with up to 2 intravenous doses of PB 20 mg/kg was tolerated during LEV titration. LEV was switched to oral as soon as the infants' condition allowed. Based on clinical observation, EEG tracings (aEEG/routine EEGs), and lab data, drug safety and anticonvulsant efficacy were assessed over 12 months.In 19 newborns a single PB dose of 20 mg/kg was administered, while 3 newborns received 2 PB doses. 30 infants were seizure free under LEV at the end of the first week and 27 remained seizure free at four weeks, while EEGs markedly improved in 24 patients at 4 weeks. In 19 cases, LEV was discontinued after 2-4 weeks, while 7 infants received LEV up to 3 months. No severe adverse effects were observed.These results illustrate the safety of LEV treatment in neonatal seizures, including prematurity and suggest LEV anticonvulsant efficacy. Additional PB treatment admittedly constitutes a methodological shortcoming due to the prolonged anticonvulsive efficacy of PB. Double blind prospective controlled studies and long-term evaluation of cognitive outcome are called for.