A review of brain biorepository management and operations.

Brain biospecimen banking requires centralized resources, national networks for referral of donors, trained personnel to interact with grieving families, and scientific staff to process the biospecimens. Process development of quality control standards is needed to meet the specific requirements of emerging genomic and proteomic technologies. Attention has to be paid to agonal factors and postmortem interval, tissue processing, neuropathology review, and long-term storage. Samples of both diseased and unaffected normal tissues are required with age- and gender-matched control tissues. Data management is vital to store and retrieve quality control measures, clinical and pathologic data linked to the biospecimens. Customized solutions for managing the acquisition and long-term storage of high-quality brain and tissue biospecimens is necessary to support neuroscience research programs, biomarker discovery and genome scale technologies. Biorepositories that operate according to best-practice policies and procedures guarantee the final wish of the families who donate tissue to support neuroscience research and discovery science.

Comprehensive cellular-resolution atlas of the adult human brain.

Detailed anatomical understanding of the human brain is essential for unraveling its functional architecture, yet current reference atlases have major limitations such as lack of whole-brain coverage, relatively low image resolution, and sparse structural annotation. We present the first digital human brain atlas to incorporate neuroimaging, high-resolution histology, and chemoarchitecture across a complete adult female brain, consisting of magnetic resonance imaging (MRI), diffusion-weighted imaging (DWI), and 1,356 large-format cellular resolution (1 µm/pixel) Nissl and immunohistochemistry anatomical plates. The atlas is comprehensively annotated for 862 structures, including 117 white matter tracts and several novel cyto- and chemoarchitecturally defined structures, and these annotations were transferred onto the matching MRI dataset. Neocortical delineations were done for sulci, gyri, and modified Brodmann areas to link macroscopic anatomical and microscopic cytoarchitectural parcellations. Correlated neuroimaging and histological structural delineation allowed fine feature identification in MRI data and subsequent structural identification in MRI data from other brains. This interactive online digital atlas is integrated with existing Allen Institute for Brain Science gene expression atlases and is publicly accessible as a resource for the neuroscience community. J. Comp. Neurol. 524:3127-3481, 2016. © 2016 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc.

Translational Research in Pediatrics IV: Solid Tissue Collection and Processing.

Solid tissues are critical for child-health research. Specimens are commonly obtained at the time of biopsy/surgery or postmortem. Research tissues can also be obtained at the time of organ retrieval for donation or from tissue that would otherwise have been discarded. Navigating the ethics of solid tissue collection from children is challenging, and optimal handling practices are imperative to maximize tissue quality. Fresh biopsy/surgical specimens can be affected by a variety of factors, including age, gender, BMI, relative humidity, freeze/thaw steps, and tissue fixation solutions. Postmortem tissues are also vulnerable to agonal factors, body storage temperature, and postmortem intervals. Nonoptimal tissue handling practices result in nucleotide degradation, decreased protein stability, artificial posttranslational protein modifications, and altered lipid concentrations. Tissue pH and tryptophan levels are 2 methods to judge the quality of solid tissue collected for research purposes; however, the RNA integrity number, together with analyses of housekeeping genes, is the new standard. A comprehensive clinical data set accompanying all tissue samples is imperative. In this review, we examined: the ethical standards relating to solid tissue procurement from children; potential sources of solid tissues; optimal practices for solid tissue processing, handling, and storage; and reliable markers of solid tissue quality.

Sudden unexpected death in epilepsy: Evaluation of forensic autopsy cases.

Epilepsy is a common chronic neurological disorder characterized by seizures. Mortality is significantly increased in patients with epilepsy. Sudden unexpected death in epilepsy (SUDEP) is the most common seizure-related category of death. A retrospective study of forensic autopsy cases from 2007 to 2009 at the Office of the Chief Medical Examiner (OCME) yielded a total of 104 sudden unexpected deaths directly or indirectly caused by an epilepsy/seizure disorder in the State of Maryland. Of these deaths, 74 cases met a general accepted definition of SUDEP. The age of SUDEP individuals ranged from 14 to 63 with the majority of subjects in the ages between 21 and 50 years (58 cases, 78.4%). Males were slightly more likely than females to die of SUDEP (male:female=1.5:1 based on the rate). The onset age of epilepsy was documented in 47.3% of cases (35/74) based on investigation and medical records. Of the 35 cases, 12 subjects had early onset epilepsy (onset ages 1-15 years) and 20 subjects had duration of epilepsy for more than 10 years. The majority of deaths (61 of the 74 cases, 82.4%) were unwitnessed. Death scene investigation showed that 71 deaths (95.9%) occurred inside their residence with 50 subjects (70.4%) found either in bed or on the bedroom floor near the bed. Forty-three out of 74 cases (58.1%) showed neuropathological lesions. Per history, 50 subjects were reported as being on anti-epileptic drugs (AEDs). However, postmortem toxicological analysis revealed that only 26 subjects (35.1%) had detectable AEDs. Of the 74 cases, seizure disorder or epilepsy was listed as primary cause of death in 66 cases and the term of SUDEP as official cause of death in only 8 cases. This report focuses on the characteristics of death scene investigation and postmortem examination findings of SUDEP cases.

Trends and pattern of drug abuse deaths in Maryland teenagers.

The Office of the Chief Medical Examiner of Maryland recorded a total of 149 drug abuse deaths of teenagers aged 13-19 years between 1991 and 2006. Of these deaths, 96 (64.4%) were caused by the use of narcotic drugs only, 29 (19.5%) by both narcotics and cocaine, four (2.7%) by both narcotics and methylenedioxymethamphetamine, six (4.0%) by cocaine only, and 14 (9.4%) by volatile substances (e.g., butane, Freon, nitrous oxide, and propane). The annual death rate from drug abuse for teenagers increased from 1.4 deaths per 100,000 population in 1991 to 2.7 deaths per 100,000 population in 2006 (chi-square test for time trend, p<0.01). The increase in teenager drug abuse deaths occurred in 1999 and since has remained at a higher rate. Further analysis revealed that the increase in drug abuse deaths was attributable to a large degree to narcotic drugs, particularly heroin/morphine and methadone, and was confined to teenagers residing in the suburban and rural areas.

Sudden unexpected death due to inflammatory myofibroblastic tumor of the heart: a case report and review of the literature.

Inflammatory myofibroblastic tumor (IMT) or inflammatory pseudotumor is a rare primary cardiac tumor that may result in sudden death. We report a sudden unexpected death due to occlusion of the coronary arteries by IMT arising from the left coronary cusp of the aortic valve. An 8-year-old child suddenly woke up from his sleep with complaint of severe chest pain to his parents, and shortly he became unresponsive. He expired 40 min later in the hospital despite resuscitation efforts. The postmortem examination revealed a 2.5 × 2 × 1-cm mass composed of multiple entangled slender cylindrical fronts, filling the coronary sinus and obstructing the coronary ostia. The patient had complained of recurrent chest pains about 2 weeks prior to his death. Echocardiogram was conducted on the patient but did not recognize the mass. Histological examination of the mass established the diagnosis of primary cardiac IMT. The detailed pathological findings are described. In addition, the literature is reviewed, and pathogenesis, clinical presentation, and the importance of forensic autopsy examination are discussed.

Abundant quantitative trait loci exist for DNA methylation and gene expression in human brain.

A fundamental challenge in the post-genome era is to understand and annotate the consequences of genetic variation, particularly within the context of human tissues. We present a set of integrated experiments that investigate the effects of common genetic variability on DNA methylation and mRNA expression in four human brain regions each from 150 individuals (600 samples total). We find an abundance of genetic cis regulation of mRNA expression and show for the first time abundant quantitative trait loci for DNA CpG methylation across the genome. We show peak enrichment for cis expression QTLs to be approximately 68,000 bp away from individual transcription start sites; however, the peak enrichment for cis CpG methylation QTLs is located much closer, only 45 bp from the CpG site in question. We observe that the largest magnitude quantitative trait loci occur across distinct brain tissues. Our analyses reveal that CpG methylation quantitative trait loci are more likely to occur for CpG sites outside of islands. Lastly, we show that while we can observe individual QTLs that appear to affect both the level of a transcript and a physically close CpG methylation site, these are quite rare. We believe these data, which we have made publicly available, will provide a critical step toward understanding the biological effects of genetic variation.

Autocrine activation of neuronal NMDA receptors by aspartate mediates dopamine- and cAMP-induced CREB-dependent gene transcription.

cAMP can stimulate the transcription of many activity-dependent genes via activation of the transcription factor, cAMP response element-binding protein (CREB). However, in mouse cortical neuron cultures, prior to synaptogenesis, neither cAMP nor dopamine, which acts via cAMP, stimulated CREB-dependent gene transcription when NR2B-containing NMDA receptors (NMDARs) were blocked. Stimulation of transcription by cAMP was potentiated by inhibitors of excitatory amino acid uptake, suggesting a role for extracellular glutamate or aspartate in cAMP-induced transcription. Aspartate was identified as the extracellular messenger: enzymatic scavenging of l-aspartate, but not glutamate, blocked stimulation of CREB-dependent gene transcription by cAMP; moreover, cAMP induced aspartate but not glutamate release. Together, these results suggest that cAMP acts via an autocrine or paracrine pathway to release aspartate, which activates NR2B-containing NMDARs, leading to Ca(2+) entry and activation of transcription. This cAMP/aspartate/NMDAR signaling pathway may mediate the effects of transmitters such as dopamine on axon growth and synaptogenesis in developing neurons or on synaptic plasticity in mature neural networks.

Direct measurement of oxidative metabolism in the living brain by microdialysis: a review.

This review summarizes microdialysis studies that address the question of which compounds serve as energy sources in the brain. Microdialysis was used to introduce 14C-labeled glucose, lactate, pyruvate, glutamate, glutamine, and acetate into the interstitial fluid of the brain to observe their metabolism to 14CO2. Although glucose uptake from the systemic system supplies the carbon source for these compounds, compounds synthesized from glucose by the brain are subject to recycling including complete metabolism to CO2. Therefore, the brain utilizes multiple compounds in its domain to provide the energy needed to fulfill its function. The physiological conditions controlling metabolism and the contribution of compartmentation into different brain regions, cell types, and subcellular spaces are still unresolved. The aconitase inhibitor fluorocitrate, with a lower inhibition threshold in glial cells, was used to identify the proportion of lactate and glucose that was oxidized in glial cells versus neurons. The fluorocitrate data suggest that glial and neuronal cells are capable of utilizing both lactate and glucose for energy metabolism.

Hormonal modulation of amino acid neurotransmitter metabolism in the arcuate nucleus of the adult female rat: a novel action of estradiol.

Morphological plasticity in response to estradiol is a hallmark of astrocytes in the arcuate nucleus. The functional consequences of these morphological changes have remained relatively unexplored. Here we report that in the arcuate nucleus estradiol significantly increased the protein levels of the two enzymes in the glutamate-glutamine cycle, glutamine synthetase and glutaminase. We further demonstrate that these estradiol-mediated changes in the enzyme protein levels may underlie functional changes in neurotransmitter availability as: 1) total glutamate concentration in the arcuate nucleus was significantly increased and 2) microdialysis revealed a significant increase in extracellular glutamate levels after a synaptic challenge in the presence of estradiol. These data implicate the glutamate-glutamine cycle in the generation and/or maintenance of glutamate and suggest that the difference in extracellular glutamate between estradiol- and oil-treated animals may be related to an increased efficiency of the cycle enzymes. In vivo enzyme activity assays revealed that the estradiol mediated increase in glutamate-glutamine cycle enzymes in the arcuate nucleus led to an increase in gamma-aminobutyric acid and is likely not related to the increase in extracellular glutamate. Thus, we have observed two-independent effects of estradiol on amino acid neurotransmission in the arcuate nucleus. These data suggest a possible functional consequence of the well-established changes in glial morphology that occur in the arcuate nucleus in the presence of estradiol and suggest the importance of neuronal-glial cooperation in the regulation of hypothalamic functions such as food intake and body weight.

Gene expression changes in the course of normal brain aging are sexually dimorphic.

Gene expression profiles were assessed in the hippocampus, entorhinal cortex, superior-frontal gyrus, and postcentral gyrus across the lifespan of 55 cognitively intact individuals aged 20-99 years. Perspectives on global gene changes that are associated with brain aging emerged, revealing two overarching concepts. First, different regions of the forebrain exhibited substantially different gene profile changes with age. For example, comparing equally powered groups, 5,029 probe sets were significantly altered with age in the superior-frontal gyrus, compared with 1,110 in the entorhinal cortex. Prominent change occurred in the sixth to seventh decades across cortical regions, suggesting that this period is a critical transition point in brain aging, particularly in males. Second, clear gender differences in brain aging were evident, suggesting that the brain undergoes sexually dimorphic changes in gene expression not only in development but also in later life. Globally across all brain regions, males showed more gene change than females. Further, Gene Ontology analysis revealed that different categories of genes were predominantly affected in males vs. females. Notably, the male brain was characterized by global decreased catabolic and anabolic capacity with aging, with down-regulated genes heavily enriched in energy production and protein synthesis/transport categories. Increased immune activation was a prominent feature of aging in both sexes, with proportionally greater activation in the female brain. These data open opportunities to explore age-dependent changes in gene expression that set the balance between neurodegeneration and compensatory mechanisms in the brain and suggest that this balance is set differently in males and females, an intriguing idea.

Oxidation of (14)C-labeled compounds perfused by microdialysis in the brains of free-moving rats.

The oxidative capacity of the brain for alternate substrates, glucose, lactate, pyruvate, acetate, glutamate, and glutamine was determined by using microdialysis to infuse (14)C-labeled compounds into the interstitial fluid of adult rat brain and by collecting the brain-generated (14)CO(2) from the dialysis eluate. All compounds were readily oxidized. The recovery of (14)CO(2) was enhanced for those compounds metabolically close to entry into the TCA cycle or known to have a low interstitial concentration. Two compounds, pyruvate and lactate, demonstrated reciprocal competition when added as nonradioactive competitors. Oxidation of two amino acids, (14)C-glutamate and (14)C-glutamine, was stimulated by the addition of nonradioactive acetate and pyruvate. alpha-Cyano-4-hydroxycinnamate decreased (14)C-lactate and (14)C-pyruvate oxidation, consistent with the transport of both compounds via a monocarboxylate transporter. The results of this in vivo study support the results of previous in vitro studies that showed that a wide range of compounds formed from glucose in the brain are also oxidized in the brain for energy production.

Effect of fluorocitrate on cerebral oxidation of lactate and glucose in freely moving rats.

Glucose is the primary carbon source to enter the adult brain for catabolic and anabolic reactions. Some studies suggest that astrocytes may metabolize glucose to lactate; the latter serving as a preferential substrate for neurons, especially during neuronal activation. The current study utilizes the aconitase inhibitor fluorocitrate to differentially inhibit oxidative metabolism in glial cells in vivo. Oxidative metabolism of 14C-lactate and 14C-glucose was monitored in vivo using microdialysis and quantitating 14CO2 in the microdialysis eluate following pulse labeling of the interstitial glucose or lactate pool. After establishing a baseline oxidation rate, fluorocitrate was added to the perfusate. Neither lactate nor glucose oxidation was affected by 5 micromol/L fluorocitrate. However, 20 and 100 micromol/L fluorocitrate reduced lactate oxidation by 55 +/- 20% and 68 +/- 12%, respectively (p < 0.05 for both). Twenty and 100 micromol/L fluorocitrate reduced 14C-glucose oxidation by 50 +/- 14% (p < 0.05) and 24 +/- 19% (ns), respectively. Addition of non-radioactive lactate to (14)C-glucose plus fluorocitrate decreased 14C-glucose oxidation by an additional 29% and 38%, respectively. These results indicate that astrocytes oxidize about 50% of the interstitial lactate and about 35% of the glucose. By subtraction, neurons metabolize a maximum of 50% of the interstitial lactate and 65% of the interstitial glucose.

Glutamate decarboxylase protects neurons against excitotoxic injury.

Although the majority of agents with antiexcitotoxic action act as glutamate receptor antagonists, enzymatic degradation of glutamate can also be neuroprotective. The very low specific activity of the mammalian form of glutamate decarboxylase (GAD), the enzyme that catalyzes the formation of gamma-aminobutyric acid (GABA) from glutamate in neurons, is likely to limit its utility as an antiglutamate neuroprotectant. In contrast, the bacterial form of GAD can be isolated with relatively high specific activity and is most active in acidic environments. We have expressed and purified GAD from Escherichia coli (bGAD) and tested the ability of the enzyme to protect against glutamate excitotoxicity. Incubation of rat hipppocampal slices with the potassium channel antagonist tetraethyl ammonium (TEA) resulted in widespread excitotoxic death of pyramidal and granule cell neurons. bGAD alone showed no significant neurotoxicity and significantly reduced excitotoxicity induced by TEA. We hypothesize that bGAD may be internalized into the synaptic vesicle compartment by nonspecific endocytosis, where both the appropriate pH and high glutamate concentrations are present. Targeting of this enzyme to the interior of synaptic vesicles may enhance its potency as a neuroprotectant against excitotoxicity.

Concentration of carbon monoxide (CO) in postmortem human tissues: effect of environmental CO exposure.

We studied how carbon monoxide (CO) is distributed within the human body through quantitation of CO concentrations in postmortem tissue samples from fatalities including possible CO exposure. Stored, frozen tissues were diced, sonicated in water, and 0.01-8.0 mg wet weight (ww) tissues were incubated with sulfosalicylic acid in CO-purged, septum-sealed vials. CO released into the headspace was quantitated by reduction gas chromatography. Mean tissue CO concentrations (pmol/mg ww) from subjects diagnosed to have no known CO exposure (control, N=14), died from fire (N=13), and CO asphyxiation (N=7), respectively, were: adipose (2;13;9), brain (3;13;65), muscle (15;97;297), heart (30;99;371), kidney (22;432;709, lung (54;690;2638), spleen (73;1366;3548), and blood (162;2238;5070). Carboxyhemoglobin concentrations were 1.4%, 25.2%, and 69.1% of total hemoglobin, respectively. We conclude that measurements of CO concentration in a variety of tissues can be used as markers for the degree of exogenous CO exposure and the identification of possible causes of death.

Receptor-mediated glutamate release from volume sensitive channels in astrocytes.

Several lines of work have shown that astrocytes release glutamate in response to receptor activation, which results in a modulation of local synaptic activity. Astrocytic glutamate release is Ca(2+)-dependent and occurs in conjunction with exocytosis of glutamate containing vesicles. However, astrocytes contain a millimolar concentration of cytosolic glutamate and express channels permeable to small anions, such as glutamate. Here, we tested the idea that astrocytes respond to receptor stimulation by dynamic changes in cell volume, resulting in volume-sensitive channel activation, and efflux of cytosolic glutamate. Confocal imaging and whole-cell recordings demonstrated that astrocytes exhibited a transient Ca(2+)-dependent cell volume increase, which activated glutamate permeable channels. HPLC analysis revealed that glutamate was released in conjunction with other amino acid osmolytes. Our observations indicate that volume-sensitive channel may constitute a previously uncharacterized target for modulation of astrocyte-neuronal interactions.

An astrocytic basis of epilepsy.

Hypersynchronous neuronal firing is a hallmark of epilepsy, but the mechanisms underlying simultaneous activation of multiple neurons remains unknown. Epileptic discharges are in part initiated by a local depolarization shift that drives groups of neurons into synchronous bursting. In an attempt to define the cellular basis for hypersynchronous bursting activity, we studied the occurrence of paroxysmal depolarization shifts after suppressing synaptic activity using tetrodotoxin (TTX) and voltage-gated Ca(2+) channel blockers. Here we report that paroxysmal depolarization shifts can be initiated by release of glutamate from extrasynaptic sources or by photolysis of caged Ca(2+) in astrocytes. Two-photon imaging of live exposed cortex showed that several antiepileptic agents, including valproate, gabapentin and phenytoin, reduced the ability of astrocytes to transmit Ca(2+) signaling. Our results show an unanticipated key role for astrocytes in seizure activity. As such, these findings identify astrocytes as a proximal target for the treatment of epileptic disorders.

Formation of extracellular glutamate from glutamine: exclusion of pyroglutamate as an intermediate.

A 4.6-fold increase in interstitial glutamate was observed following the reverse microdialysis of 5 mM glutamine into the rat hippocampus. Two possible mechanisms of glutamine hydrolysis were examined: (a) an enzymatic glutaminase activity and (b) a non-enzymatic mechanism. Injection of 14C-glutamine at the site of microdialysis followed by microdialysis with artificial cerebral spinal fluid allowed isolation of 14C-glutamine (63%), 14C-glutamate (14%), and a compound tentatively identified as pyroglutamate (22%). In this study, we determined if non-enzymatic pyroglutamate formation from glutamine contributed to the synthesis of glutamate. Pyroglutamate is in chemical equilibrium with glutamate, although under physiological conditions, the chemical equilibrium is strongly in the direction of pyroglutamate. In vitro stability studies indicated that 14C-glutamine and 14C-pyroglutamate are not subject to significant non-enzymatic breakdown at pH 6.5-7.5 at 37 degrees C for up to 8 h. Reverse microdialysis with 1 mM pyroglutamate did not increase interstitial glutamate levels. Following injection of 14C-pyroglutamate and microdialysis, radioactivity was recovered in 14C-pyroglutamate (88%) and 14C-glutamine (11%). Less than 1% of the radioactivity was recovered as glutamate. Our data do not support a role of pyroglutamate as an intermediate in the formation of extracellular glutamate following the infusion of glutamine. However, it confirms that pyroglutamate, a known constituent in brain, is actively metabolized in brain cells and contributes to glutamine in the interstitial space.

Activation of astrocytes in brain of conscious rats during acoustic stimulation: acetate utilization in working brain.

To evaluate the response of astrocytes in the auditory pathway to increased neuronal signaling elicited by acoustic stimulation, conscious rats were presented with a unilateral broadband click stimulus and functional activation was assessed by quantitative autoradiography using three tracers to pulse label different metabolic pools in brain: [2-14C]acetate labels the 'small' (astrocytic) glutamate pool, [1-14C]hydroxybutyrate labels the 'large' glutamate pool, and [14C]deoxyglucose, reflects overall glucose utilization (CMR(glc)) in all brain cells. CMR(glc) rose during brain activation, and increased activity of the oxidative pathway in working astrocytes during acoustic stimulation was registered with [2-14C]acetate. In contrast, the stimulation-induced increase in metabolic activity was not reflected by greater trapping of products of [1-14C]hydroxybutyrate. The [2-14C]acetate uptake coefficient in the inferior colliculus and lateral lemniscus during acoustic stimulation was 15% and 18% (p < 0.01) higher in the activated compared to contralateral hemisphere, whereas CMR(glc) in these structures rose by 66% (p < 0.01) and 42% (p < 0.05), respectively. Calculated rates of brain utilization of blood-borne acetate (CMR(acetate)) are about 15-25% of total CMR(glc) in non-stimulated tissue and 10-20% of CMR(glc) in acoustically activated structures; they range from 28 to 115% of estimated rates of glucose oxidation in astrocytes. The rise in acetate utilization during acoustic stimulation is modest compared to total CMR(glc), but astrocytic oxidative metabolism of 'minor' substrates present in blood can make a significant contribution to the overall energetics of astrocytes and astrocyte-neuron interactions in working brain.