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1.  Transcriptome In Vivo Analysis (TIVA) of spatially defined single cells in intact live mouse and human brain tissue 
Nature methods  2014;11(2):190-196.
Transcriptome profiling is an indispensable tool in advancing the understanding of single cell biology, but depends upon methods capable of isolating mRNA at the spatial resolution of a single cell. Current capture methods lack sufficient spatial resolution to isolate mRNA from individual in vivo resident cells without damaging adjacent tissue. Because of this limitation, it has been difficult to assess the influence of the microenvironment on the transcriptome of individual neurons. Here, we engineered a Transcriptome In Vivo Analysis (TIVA)-tag, which upon photoactivation enables mRNA capture from single cells in live tissue. Using the TIVA-tag in combination with RNA-seq to analyze transcriptome variance among single dispersed cells and in vivo resident mouse and human neurons, we show that the tissue microenvironment shapes the transcriptomic landscape of individual cells. The TIVA methodology provides the first noninvasive approach for capturing mRNA from single cells in their natural microenvironment.
doi:10.1038/nmeth.2804
PMCID: PMC3964595  PMID: 24412976
2.  Impaired K+ Homeostasis and Altered Electrophysiological Properties of Post-Traumatic Hippocampal Glia 
Traumatic brain injury (TBI) can be associated with memory impairment, cognitive deficits, or seizures, all of which can reflect altered hippocampal function. Whereas previous studies have focused on the involvement of neuronal loss in post-traumatic hippocampus, there has been relatively little understanding of changes in ionic homeostasis, failure of which can result in neuronal hyperexcitability and abnormal synchronization. Because glia play a crucial role in the homeostasis of the brain microenvironment, we investigated the effects of TBI on rat hippocampal glia. Using a fluid percussion injury (FPI) model and patch-clamp recordings from hippocampal slices, we have found impaired glial physiology 2 d after FPI. Electrophysiologically, we observed reduction in transient outward and inward K+ currents. To assess the functional consequences of these glial changes, field potentials and extracellular K+ activity were recorded in area CA3 during antidromic stimulation. An abnormal extracellular K+ accumulation was observed in the post-traumatic hippocampal slices, accompanied by the appearance of CA3 afterdischarges. After pharmacological blockade of excitatory synapses and of K+ inward currents, uninjured slices showed the same altered K+ accumulation in the absence of abnormal neuronal activity. We suggest that TBI causes loss of K+ conductance in hippocampal glia that results in the failure of glial K+ homeostasis, which in turn promotes abnormal neuronal function. These findings provide a new potential mechanistic link between traumatic brain injury and subsequent development of disorders such as memory loss, cognitive decline, seizures, and epilepsy.
PMCID: PMC4066407  PMID: 10479715
glial neuronal interactions; ion homeostasis; patch clamp; potassium selective microelectrodes; epilepsy; traumatic brain injury
3.  Posterior Fossa Ependymoma 
Skull Base  2001;11(3):219-223.
Images
PMCID: PMC1656854  PMID: 17167623
4.  Loss of Acid Sensing Ion Channel-1a and Bicarbonate Administration Attenuate the Severity of Traumatic Brain Injury 
PLoS ONE  2013;8(8):e72379.
Traumatic brain injury (TBI) is a common cause of morbidity and mortality in people of all ages. Following the acute mechanical insult, TBI evolves over the ensuing minutes and days. Understanding the secondary factors that contribute to TBI might suggest therapeutic strategies to reduce the long-term consequences of brain trauma. To assess secondary factors that contribute to TBI, we studied a lateral fluid percussion injury (FPI) model in mice. Following FPI, the brain cortex became acidic, consistent with data from humans following brain trauma. Administering HCO3− after FPI prevented the acidosis and reduced the extent of neurodegeneration. Because acidosis can activate acid sensing ion channels (ASICs), we also studied ASIC1a−/− mice and found reduced neurodegeneration after FPI. Both HCO3− administration and loss of ASIC1a also reduced functional deficits caused by FPI. These results suggest that FPI induces cerebral acidosis that activates ASIC channels and contributes to secondary injury in TBI. They also suggest a therapeutic strategy to attenuate the adverse consequences of TBI.
doi:10.1371/journal.pone.0072379
PMCID: PMC3753246  PMID: 23991103
5.  Phosphorylated tau/amyloid beta 1-42 ratio in ventricular cerebrospinal fluid reflects outcome in idiopathic normal pressure hydrocephalus 
Background
Idiopathic normal pressure hydrocephalus (iNPH) is a potentially reversible cause of dementia and gait disturbance that is typically treated by operative placement of a ventriculoperitoneal shunt. The outcome from shunting is variable, and some evidence suggests that the presence of comorbid Alzheimer's disease (AD) may impact shunt outcome. Evidence also suggests that AD biomarkers in cerebrospinal fluid (CSF) may predict the presence of AD. The aim of this study was to investigate the relationship between the phosphorylated tau/amyloid beta 1-42 (ptau/Aβ1-42) ratio in ventricular CSF and shunt outcome in patients with iNPH.
Methods
We conducted a prospective trial with a cohort of 39 patients with suspected iNPH. Patients were clinically and psychometrically assessed prior to and approximately 4 months after ventriculoperitoneal shunting. Lumbar and ventricular CSF obtained intraoperatively, and tissue from intraoperative cortical biopsies were analyzed for AD biomarkers. Outcome measures included performance on clinical symptom scales, supplementary gait measures, and standard psychometric tests. We investigated relationships between the ptau/Aβ1-42 ratio in ventricular CSF and cortical AD pathology, initial clinical features, shunt outcome, and lumbar CSF ptau/Aβ1-42 ratios in the patients in our cohort.
Results
We found that high ptau/Aβ1-42 ratios in ventricular CSF correlated with the presence of cortical AD pathology. At baseline, iNPH patients with ratio values most suggestive of AD presented with better gait performance but poorer cognitive performance. Patients with high ptau/Aβ1-42 ratios also showed a less robust response to shunting on both gait and cognitive measures. Finally, in a subset of 18 patients who also underwent lumbar puncture, ventricular CSF ratios were significantly correlated with lumbar CSF ratios.
Conclusions
Levels of AD biomarkers in CSF correlate with the presence of cortical AD pathology and predict aspects of clinical presentation in iNPH. Moreover, preliminary evidence suggests that CSF biomarkers of AD may prove useful for stratifying shunt prognosis in patients being evaluated and treated for this condition.
doi:10.1186/2045-8118-9-7
PMCID: PMC3353832  PMID: 22444461
Alzheimer's disease; Normal pressure hydrocephalus; Ventriculoperitoneal shunting; Tau; Amyloid beta 1-42; Cerebrospinal fluid
6.  Clinically silent somatotroph adenomas are common 
Objective
Somatotroph adenomas are typically recognized when they secrete GH excessively and cause acromegaly. Both ‘silent’ somatotroph adenomas (immunohistochemical evidence of GH excess without biochemical or clinical evidence) and ‘clinically silent’ somatotroph adenomas (immunohistochemical and biochemical evidence but no clinical evidence) have occasionally been reported. The relative frequency of each presentation is unknown. The goal of this study was, therefore, to determine the frequency of clinically silent somatotroph adenomas, a group that is potentially recognizable in vivo.
Design
We retrospectively identified 100 consecutive patients who had surgically excised and histologically confirmed pituitary adenomas.
Methods
Each pituitary adenoma was classified immunohistochemically by pituitary cell type. Somatotroph adenomas were further classified as ‘classic’ (obvious clinical features of acromegaly and elevated serum IGF1), ‘subtle’ (subtle clinical features of acromegaly and elevated IGF1), ‘clinically silent’ (no clinical features of acromegaly but elevated IGF1), and ‘silent’ (no clinical features of acromegaly and normal IGF1).
Results
Of the 100 consecutive pituitary adenomas, 29% were gonadotroph/glycoprotein, 24% somatotroph, 18% null cell, 15% corticotroph, 6% lactotroph, 2% thyrotroph, and 6% not classifiable. Of the 24 patients with somatotroph adenomas, classic accounted for 45.8%, subtle 16.7%, clinically silent 33.3%, and silent 4.2%.
Conclusions
Clinically silent somatotroph adenomas are more common than previously appreciated, representing one-third of all somatotroph adenomas. IGF1 should be measured in all patients with a sellar mass, because identification of a mass as a somatotroph adenoma expands the therapeutic options and provides a tumor marker to monitor treatment.
doi:10.1530/EJE-11-0216
PMCID: PMC3118490  PMID: 21493729
7.  Noninvasive Measurement of Cerebral Blood Flow and Blood Oxygenation Using Near-Infrared and Diffuse Correlation Spectroscopies in Critically Brain-Injured Adults 
Neurocritical care  2010;12(2):173-180.
Background
This study assesses the utility of a hybrid optical instrument for noninvasive transcranial monitoring in the neurointensive care unit. The instrument is based on diffuse correlation spectroscopy (DCS) for measurement of cerebral blood flow (CBF), and near-infrared spectroscopy (NIRS) for measurement of oxy- and deoxy-hemoglobin concentration. DCS/NIRS measurements of CBF and oxygenation from frontal lobes are compared with concurrent xenon-enhanced computed tomography (XeCT) in patients during induced blood pressure changes and carbon dioxide arterial partial pressure variation.
Methods
Seven neurocritical care patients were included in the study. Relative CBF measured by DCS (rCBFDCS), and changes in oxy-hemoglobin (ΔHbO2), deoxy-hemoglobin (ΔHb), and total hemoglobin concentration (ΔTHC), measured by NIRS, were continuously monitored throughout XeCT during a baseline scan and a scan after intervention. CBF from XeCT regions-of-interest (ROIs) under the optical probes were used to calculate relative XeCT CBF (rCBFXeCT) and were then compared to rCBFDCS. Spearman’s rank coefficients were employed to test for associations between rCBFDCS and rCBFXeCT, as well as between rCBF from both modalities and NIRS parameters.
Results
rCBFDCS and rCBFXeCT showed good correlation (rs = 0.73, P = 0.010) across the patient cohort. Moderate correlations between rCBFDCS and ΔHbO2/ΔTHC were also observed. Both NIRS and DCS distinguished the effects of xenon inhalation on CBF, which varied among the patients.
Conclusions
DCS measurements of CBF and NIRS measurements of tissue blood oxygenation were successfully obtained in neurocritical care patients. The potential for DCS to provide continuous, noninvasive bedside monitoring for the purpose of CBF management and individualized care is demonstrated.
doi:10.1007/s12028-009-9305-x
PMCID: PMC2844468  PMID: 19908166
Near-infrared spectroscopy; Diffuse correlation spectroscopy; Cerebral blood flow; Xenon CT; Neurocritical care
8.  Harvested human neurons engineered as live nervous tissue constructs: implications for transplantation 
Journal of neurosurgery  2008;108(2):343-347.
Object
Although neuron transplantation to repair the nervous system has shown promise in animal models, there are few practical sources of viable neurons for clinical application and insufficient approaches to bridge extensive nerve damage in patients. Therefore, the authors sought a clinically relevant source of neurons that could be engineered into transplantable nervous tissue constructs. The authors chose to evaluate human dorsal root ganglion (DRG) neurons due to their robustness in culture.
Methods
Cervical DRGs were harvested from 16 live patients following elective ganglionectomies, and thoracic DRGs were harvested from 4 organ donor patients. Following harvest, the DRGs were digested in a dispase–collagenase treatment to dissociate neurons for culture. In addition, dissociated human DRG neurons were placed in a specially designed axon expansion chamber that induces continuous mechanical tension on axon fascicles spanning 2 populations of neurons originally plated ~ 100 μm apart.
Results
The adult human DRG neurons, positively identified by neuronal markers, survived at least 3 months in culture while maintaining the ability to generate action potentials. Stretch-growth of axon fascicles in the expansion chamber occurred at the rate of 1 mm/day to a length of 1 cm, creating the first engineered living human nervous tissue constructs.
Conclusions
These data demonstrate the promise of adult human DRG neurons as an alternative transplant material due to their availability, viability, and capacity to be engineered. Also, these data show the feasibility of harvesting DRGs from living patients as a source of neurons for autologous transplant as well as from organ donors to serve as an allograft source of neurons.
doi:10.3171/JNS/2008/108/2/0343
PMCID: PMC3014262  PMID: 18240932
axon elongation; axon stretch growth; nervous tissue construct; peripheral nerve injury repair; spinal cord injury repair; tissue engineering
9.  Lack of Shunt Response in Suspected Idiopathic Normal Pressure Hydrocephalus with Alzheimer Disease Pathology 
Annals of neurology  2010;68(4):535-540.
To determine the impact of cortical Alzheimer disease pathology on shunt responsiveness in individuals treated for idiopathic normal pressure hydrocephalus (iNPH), 37 patients clinically diagnosed with iNPH participated in a prospective study in which performance on neurologic, psychometric, and gait measures before and 4 months after shunting was correlated with amyloid β plaques, neuritic plaques, and neurofibrillary tangles observed in cortical biopsies obtained during shunt insertion. No complications resulted from biopsy acquisition. Moderate to severe pathology was associated with worse baseline cognitive performance and diminished postoperative improvement on NPH symptom severity scales, gait measures, and cognitive instruments compared to patients lacking pathology.
doi:10.1002/ana.22015
PMCID: PMC2964442  PMID: 20687117
10.  A Panel of Neuron-Enriched Proteins as Markers for Traumatic Brain Injury in Humans 
Journal of Neurotrauma  2009;26(11):1867-1877.
Abstract
Surrogate markers have enormous potential for contributing to the diagnosis, prognosis, and therapeutic evaluation of acute brain damage, but extensive prior study of individual candidates has not yielded a biomarker in widespread clinical practice. We hypothesize that a panel of neuron-enriched proteins measurable in cerebrospinal fluid (CSF) and blood should vastly improve clinical evaluation and therapeutic management of acute brain injuries. Previously, we developed such a panel based initially on the study of protein release from degenerating cultured neurons, and subsequently on rodent models of traumatic brain injury (TBI) and ischemia, consisting of 14-3-3β, 14-3-3ζ, three distinct phosphoforms of neurofilament H, ubiquitin hydrolase L1, neuron-specific enolase, α-spectrin, and three calpain- and caspase-derived fragments of α-spectrin. In the present study, this panel of 11 proteins was evaluated as CSF and serum biomarkers for severe TBI in humans. By quantitative Western blotting and sandwich immunoassays, the CSF protein levels were near or below the limit of detection in pre-surgical and most normal pressure hydrocephalus (NPH) controls, but following TBI nine of the 11 were routinely elevated in CSF. Whereas different markers peaked coordinately, the time to peak varied across TBI cases from 24–96 h post-injury. In serum, TBI increased all four members of the marker panel for which sandwich immunoassays are currently available: a calpain-derived NH2-terminal α-spectrin fragment and the three neurofilament H phosphoforms. Our results identify neuron-enriched proteins that may serve as a panel of CSF and blood surrogate markers for the minimally invasive detection, management, mechanistic, and therapeutic evaluation of human TBI.
doi:10.1089/neu.2009.0882
PMCID: PMC2822802  PMID: 19811094
14-3-3; biomarker panel; calpain; necrosis; neurofilament; traumatic brain injury; UCH-L1
11.  The Relationship among Hypoxia, Proliferation, and Outcome in Patients with De Novo Glioblastoma: A Pilot Study1 
Translational Oncology  2010;3(3):160-169.
The hypoxia and proliferation index increase with grade in human glial tumors, but there is no agreement whether either has prognostic importance in glioblastomas. We evaluated these end points individually and together in 16 de novo human glioblastomas using antibodies against the 2-nitroimidazole hypoxia detection agent EF5 and the proliferation detection agent Ki-67. Frozen tumor tissue sections were fluorescence-stained for nuclei (Hoechst 33342), hypoxia (anti-EF5 antibodies), and proliferation (anti-Ki-67 antibodies). EF5 binding adjacent to Ki-67+ cells, overall EF5 binding, the ratio of these values, and the proliferation index were evaluated. Patients were classified using recursive partitioning analysis and followed up until recurrence and/or death. Recursive partitioning analysis was statistically significant for survival (P = .0026). Overall EF5 binding, EF5 binding near Ki-67+ cells, and proliferation index did not predict recurrence. Two additional survival analyses based on ratios of the overall EF5 binding to EF5 binding near Ki-67+ cells were performed. High and low ratio values were determined by two cutoff points: (a) the 50% value for the ratio [EF5/Ki-67Binding]/[Tumorbinding] = RatioEF5 50% and (b) the median EF5 value (75.6%) of the ratio [EF5/Ki-67Binding]/[Tumorbinding] = Ratiopatients median. On the basis of the RatioEF5 50%, recurrence (P = .0074) and survival (P = .0196) could be predicted. Using the Ratiopatients median, only survival could be predicted (P = .0291). In summary, patients had a worse prognosis if the [EF5/Ki-67Binding]/[Tumorbinding] ratio was high. A hypothesis for the mechanisms and translational significance of these findings is discussed.
PMCID: PMC2887645  PMID: 20563257
12.  Cognitive outcome following brain injury and treatment with an inhibitor of Nogo-A in association with an attenuated downregulation of hippocampal growth-associated protein-43 expression 
Journal of neurosurgery  2007;107(4):844-853.
Object
Central nervous system axons regenerate poorly after traumatic brain injury (TBI), partly due to inhibitors such as the protein Nogo-A present in myelin. The authors evaluated the efficacy of anti–Nogo-A monoclonal antibody (mAb) 7B12 administration on the neurobehavioral and cognitive outcome of rats following lateral fluid-percussion brain injury, characterized the penetration of the 7B12 or control antibodies into target brain regions, and evaluated the effects of Nogo-A inhibition on hemispheric tissue loss and sprouting of uninjured motor tracts in the cervical cord. To elucidate a potential molecular response to Nogo-A inhibition, we evaluated the effects of 7B12 on hippocampal GAP-43 expression.
Methods
Beginning 24 hours after lateral fluid-percussion brain injury or sham injury in rats, the mAb 7B12 or control antibody was infused intracrebroventricularly over 14 days, and behavior was assessed over 4 weeks.
Results
Immunoreactivity for 7B12 or immunoglobulin G was detected in widespread brain regions at 1 and 3 weeks postinjury. The brain-injured animals treated with 7B12 showed improvement in cognitive function (p < 0.05) at 4 weeks but no improvement in neurological motor function from 1 to 4 weeks postinjury compared with brain-injured, vehicle-treated controls. The enhanced cognitive function following inhibition of Nogo-A was correlated with an attenuated postinjury downregulation of hippocampal GAP-43 expression (p < 0.05).
Conclusions
Increased GAP-43 expression may be a novel molecular mechanism of the enhanced cognitive recovery mediated by Nogo-A inhibition after TBI in rats.
PMCID: PMC2366808  PMID: 17937233
cognition; GAP-43; neurological motor deficits; Nogo-A; regeneration; traumatic brain injury
13.  Acute Cognitive Impairment After Lateral Fluid Percussion Brain Injury Recovers by One Month: Evaluation by Conditioned Fear Response 
Behavioural brain research  2006;177(2):347-357.
Conditioned fear associates a contextual environment and cue stimulus to a foot shock in a single training trial, where fear expressed to the trained context or cue indicates cognitive performance. Lesion, aspiration or inactivation of the hippocampus and amygdala impair conditioned fear to the trained context and cue, respectively. Moreover, only bilateral experimental manipulations, in contrast to unilateral, abolish cognitive performance.
In a model of unilateral brain injury, we sought to test whether a single lateral fluid percussion brain injury impairs cognitive performance in conditioned fear. Brain-injured mice were evaluated for anterograde cognitive deficits, with the hypothesis that acute injury-induced impairments improve over time. Male C57BL/6J mice were brain-injured, trained at five or 27 days post-injury, and tested 48 hours later for recall of the association between the conditioned stimuli (trained context or cue) and the unconditioned stimulus (foot shock) by quantifying fear-associated freezing behavior. A significant anterograde hippocampal-dependent cognitive deficit was observed at seven days in brain-injured compared to sham. Cued fear conditioning could not detect amygdala-dependent cognitive deficits after injury and stereological estimation of amygdala neuron number corroborated this finding. The absence of injury-related freezing in a novel context substantiated injury-induced hippocampal-dependent cognitive dysfunction, rather than generalized fear. Variations in the training and testing paradigms demonstrated a cognitive deficit in consolidation, rather than acquisition or recall. By one month post-injury, cognitive function recovered in brain-injured mice. Hence, the acute injury-induced cognitive impairment may persist while transient pathophysiological sequelae are underway, and improve as global dysfunction subsides.
doi:10.1016/j.bbr.2006.11.014
PMCID: PMC1851906  PMID: 17169443
Head Injury; TBI; Amygdala; Hippocampus; Mouse; stereology; disector; fractionator

Results 1-13 (13)