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1.  Potential for treatment of severe autism in tuberous sclerosis complex 
The Food and Drug Administration (FDA) has approved two mechanism-based treatments for tuberous sclerosis complex (TSC)-everolimus and vigabatrin. However, these treatments have not been systematically studied in individuals with TSC and severe autism. The aim of this review is to identify the clinical features of severe autism in TSC, applicable preclinical models, and potential barriers that may warrant strategic planning in the design phase of clinical trial development. A comprehensive search strategy was formed and searched across PubMed, Embase and SCOPUS from their inception to 2/21/12, 3/16/12, and 3/12/12 respectively. After the final search date, relevant, updated articles were selected from PubMed abstracts generated electronically and emailed daily from PubMed. The references of selected articles were searched, and relevant articles were selected. A search of was completed using the search term “TSC” and “tuberous sclerosis complex”. Autism has been reported in as many as 60% of individuals with TSC; however, review of the literature revealed few data to support clear classification of the severity of autism in TSC. Variability was identified in the diagnostic approach, assessment of cognition, and functional outcome among the reviewed studies and case reports. Objective outcome measures were not used in many early studies; however, diffusion tensor imaging of white matter, neurophysiologic variability in infantile spasms, and cortical tuber subcategories were examined in recent studies and may be useful for objective classification of TSC in future studies. Mechanism-based treatments for TSC are currently available. However, this literature review revealed two potential barriers to successful design and implementation of clinical trials in individuals with severe autism-an unclear definition of the population and lack of validated outcome measures. Recent studies of objective outcome measures in TSC and further study of applicable preclinical models present an opportunity to overcome these barriers.
PMCID: PMC4145642  PMID: 25254170
Autism; Self-injury; Aggression; Tuberous sclerosis complex; Intellectual disability
2.  Treatment advances in neonatal neuroprotection and neurointensive care 
Lancet neurology  2011;10(4):372-382.
Knowledge of the nature, prognosis, and ways to treat brain lesions in neonatal infants has increased remarkably. Neonatal hypoxic-ischaemic encephalopathy (HIE) in term infants, mirrors a progressive cascade of excito-oxidative events that unfold in the brain after an asphyxial insult. In the laboratory, this cascade can be blocked to protect brain tissue through the process of neuroprotection. However, proof of a clinical effect was lacking until the publication of three positive randomised controlled trials of moderate hypothermia for term infants with HIE. These results have greatly improved treatment prospects for babies with asphyxia and altered understanding of the theory of neuroprotection. The studies show that moderate hypothermia within 6 h of asphyxia improves survival without cerebral palsy or other disability by about 40% and reduces death or neurological disability by nearly 30%. The search is on to discover adjuvant treatments that can further enhance the effects of hypothermia.
PMCID: PMC3757153  PMID: 21435600
3.  Injury to the Preterm Brain and Cerebral Palsy: Clinical Aspects, Molecular Mechanisms, Unanswered Questions, and Future Research Directions 
Journal of child neurology  2009;24(9):1064-1084.
Cerebral palsy will affect nearly 10% of the 60,000 very-low-birth-weight infants born in the United States in the next year, and an even greater percentage will display some form of permanent neurological impairment resulting from injury to the preterm brain. The 2008 Neurobiology of Disease in Children Symposium, held in conjunction with the 37th annual meeting of the Child Neurology Society, aimed to define current knowledge and to develop specific aims for future clinical, translational, and fundamental science. A complex interplay of both destructive and developmental forces is responsible for injury to the preterm brain. Advances in imaging and histology have implicated a variety of cell types, though pre-oligodendrocyte injury remains the focus. Research into different mechanisms of injury is facilitating new neuroprotective and rehabilitative interventions. A cooperative effort is necessary to translate basic research findings into clinically effective therapies and better care for these children.
PMCID: PMC3695641  PMID: 19745084
cerebral palsy; molecular mechanisms; translational research
4.  Dendrimer Brain Uptake and Targeted Therapy for Brain Injury in a Large Animal Model of Hypothermic Circulatory Arrest 
ACS Nano  2014;8(3):2134-2147.
Treatment of brain injury following circulatory arrest is a challenging health issue with no viable therapeutic options. Based on studies in a clinically relevant large animal (canine) model of hypothermic circulatory arrest (HCA)-induced brain injury, neuroinflammation and excitotoxicity have been identified as key players in mediating the brain injury after HCA. Therapy with large doses of valproic acid (VPA) showed some neuroprotection but was associated with adverse side effects. For the first time in a large animal model, we explored whether systemically administered polyamidoamine (PAMAM) dendrimers could be effective in reaching target cells in the brain and deliver therapeutics. We showed that, upon systemic administration, hydroxyl-terminated PAMAM dendrimers are taken up in the brain of injured animals and selectively localize in the injured neurons and microglia in the brain. The biodistribution in other major organs was similar to that seen in small animal models. We studied systemic dendrimer–drug combination therapy with two clinically approved drugs, N-acetyl cysteine (NAC) (attenuating neuroinflammation) and valproic acid (attenuating excitotoxicity), building on positive outcomes in a rabbit model of perinatal brain injury. We prepared and characterized dendrimer-NAC (D-NAC) and dendrimer-VPA (D-VPA) conjugates in multigram quantities. A glutathione-sensitive linker to enable for fast intracellular release. In preliminary efficacy studies, combination therapy with D-NAC and D-VPA showed promise in this large animal model, producing 24 h neurological deficit score improvements comparable to high dose combination therapy with VPA and NAC, or free VPA, but at one-tenth the dose, while significantly reducing the adverse side effects. Since adverse side effects of drugs are exaggerated in HCA, the reduced side effects with dendrimer conjugates and suggestions of neuroprotection offer promise for these nanoscale drug delivery systems.
PMCID: PMC4004292  PMID: 24499315
PAMAM dendrimers; cardiac arrest; canine model; brain injury; biodistribution; combination therapies; neuroinflammation; cytotoxicity; valproic acid; N-acetylcysteine
5.  Temporal and regional alterations in NMDA receptor expression in Mecp2-null mice 
Our previous postmortem study of girls with Rett Syndrome (RTT), a development disorder caused by MECP2 mutations, found increases in the density of NMDA receptors in the prefrontal cortex of 2–8 year-old girls, while girls older than 10 years had reductions in NMDA receptors compared to age matched controls (Blue et al., 1999b). Using [3H]-CGP to label NMDA type glutamate receptors in 2 and 7 week old wildtype (WT), Mecp2-null and Mecp2-heterozygous (HET) mice (Bird model), we found that frontal areas of the brain also exhibited a bimodal pattern in NMDA expression, with increased densities of NMDA receptors in Mecp2-null mice at 2 weeks of age, but decreased densities at 7 weeks of age. Visual cortex showed a similar pattern, while other cortical regions only exhibited changes in NMDA receptor densities at 2 weeks (retrosplenial granular) or 7 weeks (somatosensory). In thalamus of null mice, NMDA receptors were increased at 2 and 7 weeks. No significant differences in density were found between HET and WT mice at both ages. Western blots for NMDAR1 expression in frontal brain showed higher levels of expression in Mecp2-null mice at two weeks of age, but not at 1 or 7 weeks of age. Our mouse data support the notion that deficient MeCP2 function is the primary cause of the NMDA receptor changes we observed in RTT. Furthermore, the findings of regional and temporal differences in NMDA expression illustrate the importance of age and brain region in evaluating different genotypes of mice.
PMCID: PMC4122218  PMID: 21901842
Rett syndrome; NMDA; Mouse models; Development
6.  Hypoxic Ischemic Encephalopathy in the Term Infant 
Clinics in perinatology  2009;36(4):835-vii.
Hypoxia-ischemia in the perinatal period is an important cause of cerebral palsy and associated disabilities in children. There has been significant research progress in hypoxic-ischemic encephalopathy over the last two decades and many new molecular mechanisms have been identified. Despite all these advances, therapeutic interventions are still limited. In this review paper, we discuss a number of molecular pathways involved in hypoxia-ischemia, and potential therapeutic targets.
PMCID: PMC2849741  PMID: 19944838
Hypoxia ischemia; neonatal encephalopathy; apoptosis; oxidative stress; hypothermia
7.  Temporal- and Location-Specific Alterations of the GABA Recycling System in Mecp2 KO Mouse Brains 
Rett syndrome (RTT), associated with mutations in methyl-CpG-binding protein 2 (Mecp2), is linked to diverse neurological symptoms such as seizures, motor disabilities, and cognitive impairments. An altered GABAergic system has been proposed as one of many underlying pathologies of progressive neurodegeneration in several RTT studies. This study for the first time investigated the temporal- and location-specific alterations in the expression of γ-amino butyric acid (GABA) transporter 1 (GAT-1), vesicular GABA transporter (vGAT), and glutamic acid decarboxylase 67kD (GAD67) in wild type (WT) and knockout (KO) mice in the Mecp2tm1.1Bird/y mouse model of RTT. Immunohistochemistry (IHC) co-labeling of GAT-1 with vGAT identified GABAergic synapses that were quantitated for mid-sagittal sections in the frontal cortex (FC), hippocampal dentate gyrus (DG), and striatum (Str). An age-dependent increase in the expression of synaptic GABA transporters, GAT-1, and vGAT, was observed in the FC and DG in WT brains. Mecp2 KO mice showed a significant alteration in this temporal profile that was location-specific, only in the FC. GAD67-positive cell densities also showed an age-dependent increase in the FC, but a decrease in the DG in WT mice. However, these densities were not significantly altered in the KO mice in the regions examined in this study. Therefore, the significant location-specific downregulation of synaptic GABA transporters in Mecp2 KO brains with unaltered densities of GAD67-positive interneurons may highlight the location-specific synaptic pathophysiology in this model of RTT.
PMCID: PMC3981570  PMID: 24737935
Rett syndrome; MeCP2 mutation; GABAergic neurons; synapse; lipofuscin; GAT-1
Translational stroke research  2013;4(2):137-148.
Perinatal brain injuries are a leading cause of cerebral palsy worldwide. The potential of stem cell therapy to prevent or reduce these impairments has been widely discussed within the medical and scientific communities and an increasing amount of research is being conducted in this field. Animal studies support the idea that a number of stem cells types, including cord blood and mesenchymal stem cells have a neuroprotective effect in neonatal hypoxia-ischemia. Both these cell types are readily available in a clinical setting. The mechanisms of action appear to be diverse, including immunomodulation, activation of endogenous stem cells, release of growth factors, and anti-apoptotic effects. Here, we review the different types of stem cells and progenitor cells that are potential candidates for therapeutic strategies in perinatal brain injuries, and summarize recent preclinical and clinical studies.
PMCID: PMC3692557  PMID: 23814628
Stem Cell; Cerebral Palsy; neonate; brain injury; hypoxia ischemia
9.  Ischemia Induced Neuroinflammation is Associated with Disrupted Development of Oligodendrocyte Progenitors in a Model of Periventricular Leukomalacia 
Developmental neuroscience  2013;35(0):182-196.
Microglial activation in crossing white matter tracts is a hallmark of noncystic periventricular leukomalacia (PVL), the leading pathology underlying cerebral palsy in prematurely born infants. Recent studies indicate that neuroinflammation within an early time-window can produce long-lasting defects in oligodendroglial maturation, myelination-deficit, as well as disruption of transcription factors important in oligodendroglial maturation. We recently reported an ischemic mouse model of PVL, induced by unilateral neonatal carotid artery ligation, leading to selective long lasting bilateral myelination deficits, ipsilateral thinning of the corpus callosum, ventriculomegaly, as well as evidence of axonopathy.
Here, we report that permanent unilateral carotid ligation on postnatal day 5 (P5) in CD-1 mice induces an inflammatory response, as defined by microglial activation and recruitment, as well as significant changes in cytokine expression (increased IL-1b, IL-6, TGF-b1, and TNF-a) following ischemia. Transient reduction in counts of oligodendrocyte progenitor cells (OPCs) at 24 and 48 hours post-ischemia, a shift in OPC cell size and morphology towards the more immature form, as well as likely migration of OPCs were found. These OPC changes were topographically associated with areas showing microglial activation, and OPC counts negatively correlated with increased microglial staining.
The presented data shows a striking neuroinflammatory response in an ischemia-induced model of PVL, associated with oligodendroglial injury. Future studies modulating the neuroinflammatory response in this model, may contribute to a better understanding of the interaction between microglia and OPCs in PVL and open opportunities for future therapies.
PMCID: PMC3764456  PMID: 23445614
Infants; inflammation; ischemia; microglia; neonatal; oligodendrocyte progenitor; white matter
10.  Sex differences in mitochondrial biogenesis determine neuronal death and survival in response to oxygen glucose deprivation and reoxygenation 
BMC Neuroscience  2014;15:9.
Mitochondrial dysfunction has been linked to neuronal death and a wide array of neurodegenerative diseases. Previously, we have shown sex differences in mitochondria-mediated cell death pathways following hypoxia-ischemia. However, the role of mitochondrial biogenesis in hypoxic-ischemic brain injury between male vs. female has not been studied yet.
Primary cerebellar granule neurons (CGNs), isolated from P7 male and female mice (CD-1) segregated based on visual inspection of sex, were exposed to 2 h of oxygen glucose deprivation (OGD) followed by 6–24 h of reoxygenation (Reox). Mitochondrial membrane potential (ΔΨm) and cellular ATP levels were reduced significantly in XX CGNs as compared to XY CGNs. Mitochondrial DNA (mtDNA) content was increased (>2-fold) at 2 h OGD in XY CGNs and remained increased up to 24 h of Reox compared to XX neurons and normoxia controls. The expression of mitochondrial transcription factor A (Tfam), the nuclear respiratory factor-1 (NRF-1) and the peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), a master regulator of mitochondrial biogenesis, were up-regulated (2-fold, ***p < 0.001) in XY CGNs but slightly reduced or remained unchanged in XX neurons. Similarly, the TFAM and PGC-1α protein levels and the mitochondrial proteins HSP60 and COXIV were increased in XY neurons only. Supportively, a balanced stimulation of fusion (Mfn 1and Mfn 2) and fission (Fis 1 and Drp 1) genes and enhanced formation of donut-shaped mitochondria were observed in XY CGNs vs. XX neurons (**p < 0.01).
Our results demonstrate that OGD/Reox alters mitochondrial biogenesis and morphological changes in a sex-specific way, influencing neuronal injury/survival differently in both sexes.
PMCID: PMC3898007  PMID: 24410996
Hypoxia-ischemia; Mitochondrial DNA; Mitochondrial fusion and fission; Donut mitochondria; Sexual dimorphism
11.  Twenty-four hour quantitative-EEG and in-vivo glutamate biosensor detects activity and circadian rhythm dependent biomarkers of pathogenesis in Mecp2 null mice 
Mutations in the X-linked gene encoding methyl-CpG-binding protein 2 (Mecp2) cause most cases of Rett syndrome (RTT). Currently there is no cure for RTT. Abnormal EEGs are found in 100% of RTT cases and are associated with severe sleep dysfunction, the cause of which is not well understood. Mice deficient in MeCP2 protein have been studied and characterized for their neuropathological and behavioral deficits to better understand RTT. With the goal to study the non-ictal EEG correlates in symptomatic Mecp2 KO mice (Mecp2tm1.1Bird/y), and determine novel EEG biomarkers of their reported progressive neurodegeneration, we used 24 h video-EEG/EMG with synchronous in-vivo cortical glutamate biosensor in the frontal cortex. We scored the EEG for activity states and spectral analysis was performed to evaluate correlations to the synchronous extracellular glutamate fluctuations underlying Mecp2 inactivation as compared to WT. Significant alterations in sleep structure due to dark cycle-specific long wake states and poor quality of slow-wave sleep were associated with a significant increase in glutamate loads per activity cycle. The dynamics of the activity-state-dependent physiological rise and fall of glutamate indicative of glutamate homeostasis were significantly altered in the KO mice. Colorimetric quantitation of absolute glutamate levels in frontal cortex also indicated the presence of significantly higher levels in KO. This study for the first time found evidence of uncompensated sleep deprivation-like EEG biomarkers that were associated with glutamate homeostatic dysfunction in the Mecp2 KO mice.
PMCID: PMC4072927  PMID: 25018705
Mecp2; sleep structure; glutamate; biomarkers; Rett syndrome
12.  Nanomedicine in cerebral palsy 
Cerebral palsy is a chronic childhood disorder that can have diverse etiologies. Injury to the developing brain that occurs either in utero or soon after birth can result in the motor, sensory, and cognitive deficits seen in cerebral palsy. Although the etiologies for cerebral palsy are variable, neuroinflammation plays a key role in the pathophysiology of the brain injury irrespective of the etiology. Currently, there is no effective cure for cerebral palsy. Nanomedicine offers a new frontier in the development of therapies for prevention and treatment of brain injury resulting in cerebral palsy. Nanomaterials such as dendrimers provide opportunities for the targeted delivery of multiple drugs that can mitigate several pathways involved in injury and can be delivered specifically to the cells that are responsible for neuroinflammation and injury. These materials also offer the opportunity to deliver agents that would promote repair and regeneration in the brain, resulting not only in attenuation of injury, but also enabling normal growth. In this review, the current advances in nanotechnology for treatment of brain injury are discussed with specific relevance to cerebral palsy. Future directions that would facilitate clinical translation in neonates and children are also addressed.
PMCID: PMC3818020  PMID: 24204146
dendrimer; cerebral palsy; neuroinflammation; nanoparticle; neonatal brain injury; G4OH-PAMAM
13.  Everolimus and intensive behavioral therapy in an adolescent with tuberous sclerosis complex and severe behavior☆☆☆★ 
Self-injury and aggression have been reported in individuals with TSC (tuberous sclerosis complex), yet few data exist about treatment. Everolimus, an mTOR inhibitor, has been FDA-approved for subependymal giant cell astrocytomas (SEGAs) and renal angiomyolipomas in TSC. However, clinical use of everolimus with direct, real-time observations of self-injury and aggression in an individual with TSC has not been reported.
During an inpatient admission to a neurobehavioral unit, real-time measurements of behaviors and seizures were recorded. An interdisciplinary team used these data to make treatment decisions and applied behavioral and pharmacological treatments, one at a time, in order to evaluate their effects.
Aggression and self-injury improved with applied behavioral analysis (ABA), lithium, and asenapine. Improvements in SEGA size, facial angiofibromas, seizures, and the most stable low rates of self-injury were observed during the interval of treatment with everolimus.
Mechanism-based treatments in the setting of an evidence-based behavioral and psychopharmacological intervention program may be a model with utility for characterization and treatment of individuals with severe behavior and TSC.
PMCID: PMC4150649
ABA, applied behavioral analysis; CYP3A4, cytochrome p450 3A4; FDA, Food and Drug Administration; SEGA, subependymal giant cell astrocytoma; mTOR, mammalian target of rapamycin; mTORC, mammalian target of rapamycin complex; TSC, tuberous sclerosis complex; Tuberous sclerosis complex; Self-injury; Aggression; Everolimus; Behavioral intervention and epilepsy
14.  Changing the perspective on early development of Rett syndrome 
► Our study provides new insights into the pre-regressional development of RTT. ► The pre-regression period should not be considered asymptomatic. ► Peculiarities in speech-language development are potential red flags for RTT.
We delineated the achievement of early speech-language milestones in 15 young children with Rett syndrome (MECP2 positive) in the first two years of life using retrospective video analysis. By contrast to the commonly accepted concept that these children are normal in the pre-regression period, we found markedly atypical development of speech-language capacities, suggesting a paradigm shift in the pathogenesis of Rett syndrome and a possible approach to its early detection.
PMCID: PMC3605580  PMID: 23400005
Rett syndrome; Preserved speech variant; Speech-language development; Milestones; Video analysis; Regression
15.  Cost-effective therapeutic hypothermia treatment device for hypoxic ischemic encephalopathy 
Despite recent advances in neonatal care and monitoring, asphyxia globally accounts for 23% of the 4 million annual deaths of newborns, and leads to hypoxic-ischemic encephalopathy (HIE). Occurring in five of 1000 live-born infants globally and even more in developing countries, HIE is a serious problem that causes death in 25%–50% of affected neonates and neurological disability to at least 25% of survivors. In order to prevent the damage caused by HIE, our invention provides an effective whole-body cooling of the neonates by utilizing evaporation and an endothermic reaction. Our device is composed of basic electronics, clay pots, sand, and urea-based instant cold pack powder. A larger clay pot, lined with nearly 5 cm of sand, contains a smaller pot, where the neonate will be placed for therapeutic treatment. When the sand is mixed with instant cold pack urea powder and wetted with water, the device can extract heat from inside to outside and maintain the inner pot at 17°C for more than 24 hours with monitoring by LED lights and thermistors. Using a piglet model, we confirmed that our device fits the specific parameters of therapeutic hypothermia, lowering the body temperature to 33.5°C with a 1°C margin of error. After the therapeutic hypothermia treatment, warming is regulated by adjusting the amount of water added and the location of baby inside the device. Our invention uniquely limits the amount of electricity required to power and operate the device compared with current expensive and high-tech devices available in the United States. Our device costs a maximum of 40 dollars and is simple enough to be used in neonatal intensive care units in developing countries.
PMCID: PMC3540914  PMID: 23319871
therapeutic hypothermia; evaporative cooling; hypoxic ischemic encephalopathy; birth asphyxia; neuroprotection
16.  Different effects of high- and low-dose phenobarbital on post-stroke seizure suppression and recovery in immature CD1 mice 
Epilepsy research  2011;94(3):138-148.
Neonatal stroke presents with seizures that are usually treated with phenobarbital. We hypothesized that anticonvulsants would attenuate ischemic injury, but that the dose-dependent effects of standard anticonvulsants would impact important age-dependent and injury-dependent consequences. In this study, ischemia induced by unilateral carotid ligation in postnatal day 12 (P12) CD1 mice was immediately followed by an i.p. dose of vehicle, low-dose or high-dose phenobarbital. Severity of acute behavioral seizures was scored. 5-bromo-2’-deoxyuridine (BrdU) was administered from P18-P20, behavioral testing performed, and mice perfused at P40. Atrophy quantification and counts of BrdU/NeuN-labeled cells in the dentate gyrus were performed. Blood phenobarbital concentrations were measured. 30 mg/kg phenobarbital reduced acute seizures and chronic brain injury, and restored normal weight gain and exploratory behavior. By comparison, 60 mg/kg was a less efficacious anticonvulsant, was not neuroprotective, did not restore normal weight gain, and impaired behavioral and cognitive recovery. Hippocampal neurogenesis was not different between treatment groups. These results suggest a protective effect of lower-dose phenobarbital, but a lack of this effect at higher concentrations after stroke in P12 mice.
PMCID: PMC3288256  PMID: 21481568
phenobarbital; anticonvulsant; neuroprotection; neurogenesis; behavioral testing; dose-dependence
17.  In vivo magnetization transfer MRI shows dysmyelination in an ischemic mouse model of periventricular leukomalacia 
Periventricular leukomalacia, PVL, is the leading cause of cerebral palsy in prematurely born infants, and therefore more effective interventions are required. The objective of this study was to develop an ischemic injury model of PVL in mice and to determine the feasibility of in vivo magnetization transfer (MT) magnetic resonance imaging (MRI) as a potential monitoring tool for the evaluation of disease severity and experimental therapeutics. Neonatal CD-1 mice underwent unilateral carotid artery ligation on postnatal day 5 (P5); at P60, in vivo T2-weighted (T2w) and MT-MRI were performed and correlated with postmortem histopathology. In vivo T2w MRI showed thinning of the right corpus callosum, but no significant changes in hippocampal and hemispheric volumes. Magnetization transfer MRI revealed significant white matter abnormalities in the bilateral corpus callosum and internal capsule. These quantitative MT-MRI changes correlated highly with postmortem findings of reduced myelin basic protein in bilateral white matter tracts. Ventriculomegaly and persistent astrogliosis were observed on the ligated side, along with evidence of axonopathy and fewer oligodendrocytes in the corpus callosum. We present an ischemia-induced mouse model of PVL, which has pathologic abnormalities resembling autopsy reports in infants with PVL. We further validate in vivo MRI techniques as quantitative monitoring tools that highly correlate with postmortem histopathology.
PMCID: PMC3208153  PMID: 21540870
brain ischemia; glial cells; MRI; perinatal hypoxia; white matter disease
18.  Alpha II-spectrin Breakdown Products Serve as Novel Markers of Brain Injury Severity in a Canine Model of Hypothermic Circulatory Arrest 
The Annals of thoracic surgery  2009;88(2):543-550.
The development of specific biomarkers to aid in diagnosis and prognosis of neuronal injury is of paramount importance in cardiac surgery. Alpha II-spectrin is a structural protein abundant in neurons of the central nervous system and cleaved into signature fragments by proteases involved in necrotic and apoptotic cell death. We measured cerebrospinal fluid (CSF) alpha II-spectrin breakdown products (αII-SBDP’s) in a canine model of hypothermic circulatory arrest (HCA) and cardiopulmonary bypass (CPB).
Canine subjects were exposed to either 1 hour of HCA (n=8, mean lowest tympanic temperature 18.0 ± 1.2 °C), or standard CPB (n=7). CSF samples were collected prior to treatment and 8 and 24 hours post-treatment. Using polyacrylamide gel electrophoresis and immunoblotting, SBDP’s were isolated and compared between groups using computer-assisted densitometric scanning. Necrotic versus apoptotic cell death was indexed by measuring calpain and caspase-3 cleaved αII-SBDP’s (SBDP 145+150 and SBDP 120, respectively).
Animals undergoing HCA demonstrated mild patterns of histological cellular injury and clinically detectable neurologic dysfunction. Calpain-produced αII-SBDP (150kDa+145kDa bands-necrosis) 8 hours post HCA, were significantly increased (p=0.02) as compared to levels prior to HCA and remained elevated at 24 hours post HCA. In contrast caspase-3 αII-SBDP (120kDa band-apoptosis) were not significantly increased. Animals receiving CPB did not demonstrate clinical or histological evidence of injury, with no increases in necrotic or apoptotic cellular markers.
We report the use of αII-SBDP’s as markers of neurological injury following cardiac surgery. Our analysis demonstrates that Calpain and Caspase produced αII-SBDP’s may be an important and novel marker of neurologic injury following HCA.
PMCID: PMC3412404  PMID: 19632410
Brain Injury; cardiac surgery; neuroprotection; hypothermic circulatory arrest; biomarkers
19.  Derivation of Glial Restricted Precursors from E13 mice 
This is a protocol for derivation of glial restricted precursor (GRP) cells from the spinal cord of E13 mouse fetuses. These cells are early precursors within the oligodendrocytic cell lineage. Recently, these cells have been studied as potential source for restorative therapies in white matter diseases. Periventricular leukomalacia (PVL) is the leading cause of non-genetic white matter disease in childhood and affects up to 50% of extremely premature infants. The data suggest a heightened susceptibility of the developing brain to hypoxia-ischemia, oxidative stress and excitotoxicity that selectively targets nascent white matter. Glial restricted precursors (GRP), oligodendrocyte progenitor cells (OPC) and immature oligodendrocytes (preOL) seem to be key players in the development of PVL and are the subject of continuing studies. Furthermore, previous studies have identified a subset of CNS tissue that has increased susceptibility to glutamate excitotoxicity as well as a developmental pattern to this susceptibility. Our laboratory is currently investigating the role of oligodendrocyte progenitors in PVL and use cells at the GRP stage of development. We utilize these derived GRP cells in several experimental paradigms to test their response to select stresses consistent with PVL. GRP cells can be manipulated in vitro into OPCs and preOL for transplantation experiments with mouse PVL models and in vitro models of PVL-like insults including hypoxia-ischemia. By using cultured cells and in vitro studies there would be reduced variability between experiments which facilitates interpretation of the data. Cultured cells also allows for enrichment of the GRP population while minimizing the impact of contaminating cells of non-GRP phenotype.
PMCID: PMC3399460  PMID: 22760029
Neuroscience;  Issue 64;  Physiology;  Medicine;  periventricular leukomalacia;  oligodendrocytes;  glial restricted precursors;  spinal cord;  cell culture
20.  Developmental Neuroscience Relevant to Child Neurology 
Seminars in Pediatric Neurology  2011;18(2):133-138.
PMCID: PMC3289954  PMID: 22036501
21.  Plasticity and mTOR: Towards Restoration of Impaired Synaptic Plasticity in mTOR-Related Neurogenetic Disorders 
Neural Plasticity  2012;2012:486402.
Objective. To review the recent literature on the clinical features, genetic mutations, neurobiology associated with dysregulation of mTOR (mammalian target of rapamycin), and clinical trials for tuberous sclerosis complex (TSC), neurofibromatosis-1 (NF1) and fragile X syndrome (FXS), and phosphatase and tensin homolog hamartoma syndromes (PTHS), which are neurogenetic disorders associated with abnormalities in synaptic plasticity and mTOR signaling. Methods. Pubmed and were searched using specific search strategies. Results/Conclusions. Although traditionally thought of as irreversible disorders, significant scientific progress has been made in both humans and preclinical models to understand how pathologic features of these neurogenetic disorders can be reduced or reversed. This paper revealed significant similarities among the conditions. Not only do they share features of impaired synaptic plasticity and dysregulation of mTOR, but they also share clinical features—autism, intellectual disability, cutaneous lesions, and tumors. Although scientific advances towards discovery of effective treatment in some disorders have outpaced others, progress in understanding the signaling pathways that connect the entire group indicates that the lesser known disorders will become treatable as well.
PMCID: PMC3350854  PMID: 22619737
22.  Serum Levels of Neuron-Specific UCHL1 Predict Brain Injury in a Canine Model of Hypothermic Circulatory Arrest 
Ubiquitin carboxyl-terminal esterase-L1 (UCHL1) is a protein highly selectively expressed in neurons and has been linked to neurodegenerative disease in humans. We hypothesize that UCHL1 would be an effective serum biomarker for brain injury as tested in canine models of hypothermic circulatory arrest (HCA) and cardiopulmonary bypass (CPB).
Canines were exposed to CPB (n=14), 1 hour(h) HCA (n=11), or 2h-HCA (n=20). Cerebrospinal fluid (CSF) and serum were collected at baseline, 8h, and 24h post-treatment. UCHL1 levels were measured using a sandwich enzyme-linked immunosorbent assay (ELISA). Neurological function and histopathology were scored at 24h, and UCHL1 immunoreactivity was examined at 8h.
Baseline UCHL1 protein levels in CSF and serum were similar for all groups. In serum, UCHL1 levels were elevated at 8h post-treatment for 2h-HCA subjects compared to baseline values (p<0.01), and also compared to CPB canines at 8h (p<0.01). A serum UCHL1 level above 3.9ng/(mg total protein) at 8h had the best discriminatory power for predicting functional disability. In CSF, UCHL1 was elevated in all groups at 8h post-treatment compared to baseline (p<0.01). However, UCHL1 levels in CSF remained elevated at 24h only in 2h-HCA subjects (p<0.01). Functional and histopathology scores were closely correlated (Pearson’s coefficient: 0.66; p<0.01), and were significantly worse in 2h-HCA animals.
This is the first report associating elevated serum UCHL1 with brain injury. The novel neuronal biomarker UCHL1 is increased in serum 8h after severe neurological insult in 2h-HCA animals compared with CPB animals. These results support the potential for use in cardiac surgery patients, and form the basis for clinical correlation in humans.
PMCID: PMC3177106  PMID: 21924148
Animal Model; Cardiopulmonary bypass (CPB); Biomarker; Hypothermia/circulatory arrest; Neurology/Neurologic injury
23.  Glutamate Excitotoxicity Mediates Neuronal Apoptosis After Hypothermic Circulatory Arrest 
The Annals of thoracic surgery  2010;89(2):440-445.
Prolonged hypothermic circulatory arrest results in neuronal cell death and neurologic injury. We have previously shown that hypothermic circulatory arrest causes both neuronal apoptosis and necrosis in a canine model. Inhibition of neuronal nitric oxide synthase reduced neuronal apoptosis, while glutamate receptor antagonism reduced necrosis in our model. This study was undertaken to determine whether glutamate receptor antagonism reduces nitric oxide formation and neuronal apoptosis after hypothermic circulatory arrest.
Sixteen hound dogs underwent 2 hours of circulatory arrest at 18°C and were sacrificed after 8 hours. Group 1 (n=8) was treated with MK-801, 0.75 mg/kg IV prior to arrest followed by 75 μg/kg/hr infusion. Group 2 dogs (n=8) received vehicle only. Intracerebral levels of excitatory amino acids and citrulline, an equal co-product of nitric oxide, were measured. Apoptosis, identified by H&E staining and confirmed by electron microscopy, was blindly scored from 0 (normal) to 100 (severe injury), while nick-end labeling demonstrated DNA fragmentation.
Group 1 and 2 dogs had similar intracerebral levels of glutamate. However, MK-801 significantly reduced intracerebral glycine and citrulline levels as compared to HCA controls. MK-801 significantly inhibited apoptosis (7.92 ± 7.85 vs. 62.08 ± 6.28, Group 1 vs. 2, p<0.001).
Our results showed that glutamate receptor antagonism significantly reduced nitric oxide formation and neuronal apoptosis. We provide evidence that glutamate excitotoxicity mediates neuronal apoptosis in addition to necrosis after hypothermic circulatory arrest. Clinical glutamate receptor antagonists may have therapeutic benefit in ameliorating both types of neurologic injury after hypothermic circulatory arrest.
PMCID: PMC3076934  PMID: 20103318
Animal Model; Apoptosis; Brain; Hypothermic Circulatory Arrest; Nitric Oxide
24.  Sex-specific activation of cell death signalling pathways in cerebellar granule neurons exposed to oxygen glucose deprivation followed by reoxygenation 
ASN NEURO  2011;3(2):e00056.
Neuronal death pathways following hypoxia–ischaemia are sexually dimorphic, but the underlying mechanisms are unclear. We examined cell death mechanisms during OGD (oxygen-glucose deprivation) followed by Reox (reoxygenation) in segregated male (XY) and female (XX) mouse primary CGNs (cerebellar granule neurons) that are WT (wild-type) or Parp-1 [poly(ADP-ribose) polymerase 1] KO (knockout). Exposure of CGNs to OGD (1.5 h)/Reox (7 h) caused cell death in XY and XX neurons, but cell death during Reox was greater in XX neurons. ATP levels were significantly lower after OGD/Reox in WT-XX neurons than in XY neurons; this difference was eliminated in Parp-1 KO-XX neurons. AIF (apoptosis-inducing factor) was released from mitochondria and translocated to the nucleus by 1 h exclusively in WT-XY neurons. In contrast, there was a release of Cyt C (cytochrome C) from mitochondria in WT-XX and Parp-1 KO neurons of both sexes; delayed activation of caspase 3 was observed in the same three groups. Thus deletion of Parp-1 shunted cell death towards caspase 3-dependent apoptosis. Delayed activation of caspase 8 was also observed in all groups after OGD/Reox, but was much greater in XX neurons, and caspase 8 translocated to the nucleus in XX neurons only. Caspase 8 activation may contribute to increased XX neuronal death during Reox, via caspase 3 activation. Thus, OGD/Reox induces death of XY neurons via a PARP-1-AIF-dependent mechanism, but blockade of PARP-1-AIF pathway shifts neuronal death towards a caspase-dependent mechanism. In XX neurons, OGD/Reox caused prolonged depletion of ATP and delayed activation of caspase 8 and caspase 3, culminating in greater cell death during Reox.
PMCID: PMC3072765  PMID: 21382016
apoptosis; caspase 3; caspase 8; hypoxia–ischaemia; neuronal death; sexual dimorphism; AIF, apoptosis-inducing factor; AM: acetoxymethyl ester, ; CGN, cerebellar granule neuron; Cyt C, cytochrome c; DAPI, 4′,6-diamidino-2-phenylindole; DIV 9, 9 days in vitro; HBSS, Hanks' balanced salt solution; HI, hypoxia–ischaemia; HRP, horseradish peroxidase; KO, knockout; LDH, lactate dehydrogenase; MB, mitochondrial buffer; OGD, oxygen-glucose deprivation; PI, propidium iodide; pNA, p-nitroaniline; Parp-1/PARP-1, poly(ADP-ribose) polymerase-1; Reox, reoxygenation; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling; VDAC, voltage-dependent anion channel; WT, wild-type
25.  Transcriptional Profile of Brain Injury in Hypothermic Circulatory Arrest and Cardiopulmonary Bypass 
The Annals of thoracic surgery  2010;89(6):1965-1971.
Little is known about the molecular mechanisms of neurologic complications after hypothermic circulatory arrest (HCA) with cardiopulmonary bypass (CPB). Canine genome sequencing allows profiling of genomic changes after HCA and CPB alone. We hypothesize that gene regulation will increase with increased severity of injury.
Dogs underwent 2-hour HCA at 18°C (n = 10), 1-hour HCA (n = 8), or 2-hour CPB at 32°C alone (n = 8). In each group, half were sacrificed at 8 hours and half at 24 hours after treatment. After neurologic scoring, brains were harvested for genomic analysis. Hippocampal RNA isolates were analyzed using canine oligonucleotide expression arrays containing 42,028 probes.
Consistent with prior work, dogs that underwent 2-hour HCA experienced severe neurologic injury. One hour of HCA caused intermediate clinical damage. Cardiopulmonary bypass alone yielded normal clinical scores. Cardiopulmonary bypass, 1-hour HCA, and 2-hour HCA groups historically demonstrated increasing degrees of histopathologic damage (previously published). Exploratory analysis revealed differences in significantly regulated genes (false discovery rate < 10%, absolute fold change ≥ 1.2), with increases in differential gene expression with injury severity. At 8 hours and 24 hours after insult, 2-hour HCA dogs had 502 and 1,057 genes regulated, respectively; 1-hour HCA dogs had 179 and 56 genes regulated; and CPB alone dogs had 5 and 0 genes regulated.
Our genomic profile of canine brains after HCA and CPB revealed 1-hour and 2-hour HCA induced markedly increased gene regulation, in contrast to the minimal effect of CPB alone. This adds to the body of neurologic literature supporting the safety of CPB alone and the minimal effect of CPB on a normal brain, while illuminating genomic results of both.
PMCID: PMC3031914  PMID: 20494057

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