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1.  A GSK-3β inhibitor protects against radiation necrosis in mouse brain 
To quantify the effectiveness of SB415286, a specific inhibitor of GSK-3β, as a neuroprotectant of radiation-induced, CNS (brain) necrosis in a mouse model.
Methods and Materials
Cohorts of mice were treated with SB415286 or DMSO prior to irradiation with a single 45-Gy fraction targeted to the left hemisphere (brain) using the Leksell Perfexion Gamma Knife. The onset and progression of radiation necrosis were monitored longitudinally by non-invasive in vivo, small-animal MRI, beginning 13 weeks post-irradiation. MRI-derived necrotic volumes for SB415286- and DMSO-treated mice were compared. MRI results were supported by correlative histology.
Mice treated with SB415286 showed significant protection from radiation-induced necrosis, as determined by in vivo MRI with histologic validation. MRI-derived necrotic volumes were significantly smaller at all post-irradiation time points in SB415286-treated animals. While the irradiated hemispheres of the DMSO-treated mice demonstrated many of the classic histologic features of RN, including fibrinoid vascular necrosis, vascular telangiectasia, hemorrhage, and tissue loss, the irradiated hemisphere of the SB415286-treated mice consistently showed only minimal tissue damage. These studies confirmed that treatment with a GSK-3β inhibitor dramatically reduced delayed time-to-onset necrosis in irradiated brain.
The unilateral cerebral hemispheric stereotactic radiosurgery mouse model, in concert with longitudinal MRI monitoring, provides a powerful platform for studying the onset and progression of radiation necrosis and for developing and testing new neuroprotectants. SB415286’s effectiveness as a neuroprotectant for necrosis motivates potential clinical trials of it or other GSK-3β inhibitors.
PMCID: PMC4307920  PMID: 24969790
Magnetic resonance imaging; Radiation necrosis; Neuroprotection; Small animal models; Radiotherapy
2.  A Cost-Effectiveness Analysis of First Trimester Non-Invasive Prenatal Screening for Fetal Trisomies in the United States 
PLoS ONE  2015;10(7):e0131402.
Non-invasive prenatal testing (NIPT) is a relatively new technology for diagnosis of fetal aneuploidies. NIPT is more accurate than conventional maternal serum screening (MSS) but is also more costly. Contingent NIPT may provide a cost-effective alternative to universal NIPT screening. Contingent screening used a two-stage process in which risk is assessed by MSS in the first stage and, based on a risk cutoff, high-risk pregnancies are referred for NIPT. The objective of this study was to (1) determine the optimum MSS risk cutoff for contingent NIPT and (2) compare the cost effectiveness of optimized contingent NIPT to universal NIPT and conventional MSS.
Study Design
Decision-analytic model using micro-simulation and probabilistic sensitivity analysis. We evaluated cost effectiveness from three perspectives: societal, governmental, and payer.
From a societal perspective, universal NIPT dominated both contingent NIPT and MSS. From a government and payer perspective, contingent NIPT dominated MSS. Compared to contingent NIPT, adopting a universal NIPT would cost $203,088 for each additional case detected from a government perspective and $263,922 for each additional case detected from a payer perspective.
From a societal perspective, universal NIPT is a cost-effective alternative to MSS and contingent NIPT. When viewed from narrower perspectives, contingent NIPT is less costly than universal NIPT and provides a cost-effective alternative to MSS.
PMCID: PMC4489811  PMID: 26133556
3.  Anti-VEGF antibodies mitigate the development of radiation necrosis in mouse brain 
To quantify the effectiveness of anti-VEGF antibodies (bevacizumab and B20-4.1.1) as mitigators of radiation-induced, CNS (brain) necrosis in a mouse model.
Experimental Design
Cohorts of mice were irradiated with single-fraction 50- or 60-Gy doses of radiation targeted to the left hemisphere (brain) using the Leksell Perfexion Gamma Knife. The onset and progression of radiation necrosis were monitored longitudinally by in vivo, small-animal MRI, beginning four weeks post-irradiation. MRI-derived necrotic volumes for antibody (Ab)-treated and untreated mice were compared. MRI results were supported by correlative histology.
Hematoxylin and eosin stained sections of brains from irradiated, non-Ab-treated mice confirmed profound tissue damage, including regions of fibrinoid vascular necrosis, vascular telangiectasia, hemorrhage, loss of neurons, and edema. Treatment with the murine anti-VEGF antibody B20-4.1.1 mitigated radiation-induced changes in an extraordinary, highly statistically-significant manner. The development of radiation necrosis in mice under treatment with bevacizumab (a humanized anti-VEGF antibody) was intermediate between that for B20-4.1.1-treated and non-Ab-treated animals. MRI findings were validated by histologic assessment, which confirmed that anti-VEGF-antibody treatment dramatically reduced late-onset necrosis in irradiated brain.
The single-hemispheric-irradiation mouse model, with longitudinal MRI monitoring, provides a powerful platform for studying the onset and progression of radiation necrosis and for developing and testing new therapies. The observation that anti-VEGF antibodies are effective mitigants of necrosis in our mouse model will enable a wide variety of studies aimed at dose optimization and timing and mechanism of action with direct relevance to ongoing clinical trials of bevacizumab as a treatment for radiation necrosis.
PMCID: PMC4135174  PMID: 24647570
4.  New Roles for the External Globus Pallidus in Basal Ganglia Circuits and Behavior 
The Journal of Neuroscience  2014;34(46):15178-15183.
The development of methodology to identify specific cell populations and circuits within the basal ganglia is rapidly transforming our ability to understand the function of this complex circuit. This mini-symposium highlights recent advances in delineating the organization and function of neural circuits in the external segment of the globus pallidus (GPe). Although long considered a homogeneous structure in the motor-suppressing “indirect-pathway,” the GPe consists of a number of distinct cell types and anatomical subdomains that contribute differentially to both motor and nonmotor features of behavior. Here, we integrate recent studies using techniques, such as viral tracing, transgenic mice, electrophysiology, and behavioral approaches, to create a revised framework for understanding how the GPe relates to behavior in both health and disease.
PMCID: PMC4228126  PMID: 25392486
5.  Perilesional edema in radiation necrosis reflects axonal degeneration 
Recently, we characterized a Gamma Knife® radiation necrosis mouse model with various magnetic resonance imaging (MRI) protocols to identify biomarkers useful in differentiation from tumors. Though the irradiation was focal to one hemisphere, a contralateral injury was observed that appeared to be localized in the white matter only. Interestingly, this injury was identifiable in T2-weighted images, apparent diffusion coefficient (ADC), and magnetization transfer ratio (MTR) maps, but not on post-contrast T1-weighted images. This observation of edema independent of vascular changes is akin to the perilesional edema seen in clinical radiation necrosis.
The pathology underlying the observed white-matter MRI changes was explored by performing immunohistochemistry for healthy axons and myelin. The presence of both healthy axons and myelin was reduced in the contralateral white-matter lesion.
Based on our immunohistochemical findings, the contralateral white-matter injury is most likely due to axonal degeneration.
PMCID: PMC4331301  PMID: 25636531
Radiation necrosis; White matter injury; Axonal degeneration
6.  Tracking donor RBC survival in premature infants: agreement of multiple populations of biotin-labeled RBCs with Kidd antigen mismatched RBCs 
Pediatric research  2013;74(6):689-697.
Anemia, a common condition among critically ill premature infants, is affected by red blood cell (RBC) survival (RCS). We hypothesized that transfused allogeneic Kidd antigen mismatched RBCs would demonstrate the same concurrent RCS tracking as RBCs multi-labeled at separate, discrete low densities with biotin (BioRBCs).
Allogeneic RBCs from adult donors were labeled at four biotin densities, mixed, and transfused into 17 anemic premature infants. Nine of the donors and neonates were Kidd antigen mismatched. Serial post-transfusion blood samples were assayed for up to eight weeks by flow cytometry to track the survival of the proportions of Kidd antigen mismatched and biotinylated RBCs.
Using linear mixed modeling to compare results, RCS of the three lowest BioRBC densities was similar to RCS by Kidd antigen mismatch and to one another. RCS of RBCs labeled at the highest BioRBC density was shortened.
RCS of different populations of RBCs can be tracked concurrently and reliably using the three lowest BioRBC densities. Although comparable RCS results can be achieved using Kidd antigen mismatches, BioRBCs are preferred for investigating neonatal anemias because biotin labeling of both allogeneic and autologous RBCs is possible.
PMCID: PMC3913052  PMID: 24108188
7.  Verification bias is common in cytopathology studies on diagnostic accuracy 
CytoJournal  2014;11:13.
PMCID: PMC4058898  PMID: 24987444
8.  Enhanced sphingosine-1-phosphate receptor 2 expression underlies female CNS autoimmunity susceptibility 
The Journal of Clinical Investigation  2014;124(6):2571-2584.
Multiple sclerosis (MS) is an inflammatory disease of the CNS that is characterized by BBB dysfunction and has a much higher incidence in females. Compared with other strains of mice, EAE in the SJL mouse strain models multiple features of MS, including an enhanced sensitivity of female mice to disease; however, the molecular mechanisms that underlie the sex- and strain-dependent differences in disease susceptibility have not been described. We identified sphingosine-1-phosphate receptor 2 (S1PR2) as a sex- and strain-specific, disease-modifying molecule that regulates BBB permeability by destabilizing adherens junctions. S1PR2 expression was increased in disease-susceptible regions of the CNS of both female SJL EAE mice and female patients with MS compared with their male counterparts. Pharmacological blockade or lack of S1PR2 signaling decreased EAE disease severity as the result of enhanced endothelial barrier function. Enhanced S1PR2 signaling in an in vitro BBB model altered adherens junction formation via activation of Rho/ROCK, CDC42, and caveolin endocytosis-dependent pathways, resulting in loss of apicobasal polarity and relocation of abluminal CXCL12 to vessel lumina. Furthermore, S1PR2-dependent BBB disruption and CXCL12 relocation were observed in vivo. These results identify a link between S1PR2 signaling and BBB polarity and implicate S1PR2 in sex-specific patterns of disease during CNS autoimmunity.
PMCID: PMC4089451  PMID: 24812668
9.  Comparison of multiple red cell volume methods performed concurrently in premature infants following allogeneic transfusion 
Pediatric research  2013;74(5):592-600.
Study of the pathophysiology and treatment of anemia of prematurity is facilitated by direct measurement of red cell volume (RCV) utilizing microliter quantities of blood samples. Our objective was to compare concurrent measurements of multiple direct RCV methods in infants.
Eighteen preterm infants receiving clinically-indicated transfusions had concurrent flow cytometric determinations of RCV and 24 h red blood cell (RBC) recovery based on donor-recipient differences of biotin–labeled RBCs (BioRBCs), Kidd antigen mismatched RBCs, and HbF positive (HbF+) RBCs. HPLC was also used to measure HbF and HbA protein concentrations for RCV determination.
Concurrent RCV measurements using BioRBCs (18 and 54 μg/ml), Kidd antigen, and HbF flow cytometry were not statistically different compared to RCVs measured using the reference BioRBC density (6 μg/ml). In contrast, the HbF HPLC method over estimated RCV by 45% compared to the reference method. All methods demonstrated 100% 24 h post-transfusion RBC recovery (PTR24).
Because BioRBC, Kidd antigen, and HbF flow cytometry are safe and accurate methods requiring <10 μl of patient blood to determine RCV and PTR24 in preterm infants, they can be useful in clinical and research studies of anemia and other conditions.
PMCID: PMC3907947  PMID: 24088873
10.  Role of Magnetic Resonance Imaging, Cerebrospinal Fluid, and Electroencephalogram in Diagnosis of Sporadic Creutzfeldt-Jakob Disease 
Journal of neurology  2012;260(2):498-506.
Sporadic Creutzfeldt-Jakob disease (sCJD) is a rapidly progressive dementia (RPD) that can be difficult to identify ante-mortem, with definitive diagnosis requiring tissue confirmation. We describe the clinical, magnetic resonance imaging (MRI), cerebrospinal fluid (CSF), and electroencephalogram (EEG) measures of a small cohort of 30 patients evaluated for RPD. Clinical and diagnostic measures were cross-sectionally obtained from 17 sCJD patients (15 definite, 2 probable), 13 non-prion rapidly progressive dementia patients (npRPD), and 18 unimpaired controls. In a subset of patients (9 sCJD and 9 npRPD) diffusion tensor imaging (DTI) measures [fractional anisotropy (FA), mean diffusivity (MD), axial diffusivity (AD), and radial diffusivity (RD)] were also obtained for the caudate, corpus callosum, posterior limb of the internal capsule, pulvinar, precuneus, and frontal lobe. Differences among groups were assessed by an analysis of variance. Compared to npRPD individuals, sCJD patients had cerebellar dysfunction, significantly higher CSF tau, “positive” CSF 14-3-3, and hyperintensities on diffusion weighted imaging (DWI) that met previously established imaging criteria for sCJD. EEG changes were similar for the two groups. In addition, sCJD patients had significant decreases in DTI measures (MD, AD, RD but not FA) within the caudate and pulvinar compared to either npRPD patients or unimpaired controls. Our results confirm that CSF abnormalities and MRI (especially DWI) can assist in distinguishing sCJD patients from npRPD patients. Future longitudinal studies using multiple measures (including CSF and MRI) are needed for evaluating pathological changes seen in sCJD patients.
PMCID: PMC3651672  PMID: 22968768
Creutzfeldt-Jakob disease; diffusion magnetic resonance imaging; cerebrospinal fluid
11.  Canceling actions involves a race between basal ganglia pathways 
Nature neuroscience  2013;16(8):1118-1124.
Salient cues can prompt the rapid interruption of planned actions. It has been proposed that fast, reactive behavioral inhibition involves specific basal ganglia pathways, and we tested this by comparing activity in multiple rat basal ganglia structures during performance of a stop-signal task. Subthalamic nucleus (STN) neurons showed low-latency responses to Stop cues, irrespective of whether actions were successfully canceled or not. By contrast, neurons downstream in the substantia nigra pars reticulata (SNr) responded to Stop cues only in trials with successful cancellation. Recordings and simulations together indicate that this sensorimotor gating arises from the relative timing of two distinct inputs to neurons in the SNr dorsolateral “core” subregion: cue-related excitation from STN and movement-related inhibition from striatum. Our results support race models of action cancellation, with successful stopping requiring Stop cue information to be transmitted from STN to SNr before increased striatal input creates a point of no return.
PMCID: PMC3733500  PMID: 23852117
12.  Dopamine modulation of learning and memory in the prefrontal cortex: insights from studies in primates, rodents, and birds 
In this review, we provide a brief overview over the current knowledge about the role of dopamine transmission in the prefrontal cortex during learning and memory. We discuss work in humans, monkeys, rats, and birds in order to provide a basis for comparison across species that might help identify crucial features and constraints of the dopaminergic system in executive function. Computational models of dopamine function are introduced to provide a framework for such a comparison. We also provide a brief evolutionary perspective showing that the dopaminergic system is highly preserved across mammals. Even birds, following a largely independent evolution of higher cognitive abilities, have evolved a comparable dopaminergic system. Finally, we discuss the unique advantages and challenges of using different animal models for advancing our understanding of dopamine function in the healthy and diseased brain.
PMCID: PMC4122189  PMID: 25140130
prefrontal cortex; learning and memory; dopamine receptors; executive function; working memory; neuromodulation; evolution
14.  Independent Evaluation of Computer Classification of Malignant and Benign Calcifications in Full-Field Digital Mammograms 
Academic radiology  2007;14(3):363-370.
Rationale and Objectives
To evaluate whether a computer-aided diagnosis (CADx) technique can accurately classify breast calcifications in full-field digital mammograms (FFDMs) as malignant or benign. The computer technique was developed previously on screen-film mammograms (SFMs) in which individual calcifications were identified manually. The present study evaluated the computer technique independently on a new database of FFDM images with automatic detection of the individual calcifications.
Materials and Methods
We analyzed 49 consecutive FFDM cases (19 cancers) that showed suspicious calcifications. Four mammography radiologists read softcopy mammograms retrospectively and electronically indicated the region of calcifications in each image. The computer then automatically detected the individual calcifications within the indicated region and analyzed eight features of calcification morphology and distribution to arrive at an estimated likelihood of malignancy. The radiologists entered BI-RADS assessments before and after seeing the computer results. Performance was analyzed using receiver operating characteristic (ROC) analysis.
Despite variability in radiologist-indicated regions of calcifications, the computer achieved consistently high performance taking input from the four radiologists (ROC curve area, Az, of 0.80, 0.80, 0.78, and 0.77; differences not statistically significant). Previous results showed that the computer technique achieved an Az value of 0.80 on SFMs, which improved radiologists’ performance significantly.
The computer technique appears to maintain consistently high performance classifying calcifications in FFDMs as malignant or benign without requiring substantial modification from its initial development on SFMs. The computer performance appears to be robust with respect to variations in radiologists’ input.
PMCID: PMC3654797  PMID: 17307670
computer-aided diagnosis; full-field digital mammography; diagnostic mammography; breast calcifications; breast cancer diagnosis
15.  Differential pharmacokinetic analysis of in vivo erythropoietin receptor interaction with erythropoietin and continuous erythropoietin receptor activator in sheep 
The two erythropoiesis stimulating agents (ESAs), short acting recombinant human erythropoietin (EPO) and long acting continuous erythropoietin receptor activator (CERA), have been hypothesized to share an in vivo elimination pathway that involves binding to erythropoietin receptor (EPOR) and subsequent internalization. A physiologically based recirculation model and a pharmacokinetic tracer interaction methodology (TIM) were used to compare the in vivo interaction kinetics with EPOR between the two ESAs in adult sheep. Animals treated with EPO experienced a greater EPOR up-regulation than those treated with CERA, as evidenced by an eightfold-higher initial EPOR normalized production rate constant, ksyn/R0, versus a twofold-larger EPOR degradation rate constant, kdeg. In agreement with in vitro studies, EPO had a lower in vivo equilibrium dissociation constant from EPOR than CERA (KD = 6 versus 88.4 pmol/l, respectively, p < 0.01). The internalization and/or degradation of the EPO–EPOR complex was faster than that of the CERA–EPOR complex (kint = 24 versus 2.41 h−1, respectively, p < 0.01). The adopted model enables a mechanism-based explanation for CERA’s slower elimination and greater erythropoietic activity in vivo. As predicted by the model, the slower elimination of CERA is due to: (1) less EPOR up-regulation induced by CERA administration; (2) slower binding of CERA to EPOR; and (3) reduced internalization and/or degradation rate of surface-bound CERA. Slower CERA/EPOR complex elimination explains the greater in vivo erythropoiesis reported for CERA, despite its lower affinity to EPOR. A sensitivity analysis showed that the model parameters were reliably estimated using the TIM methodology.
PMCID: PMC3616385  PMID: 21678432
erythropoietin; erythropoiesis stimulating agent (ESA); CERA; pharmacokinetics; receptor; physiological model; receptor binding; recirculation model
16.  Basal Ganglia Beta Oscillations Accompany Cue Utilization 
Neuron  2012;73(3):523-536.
Beta oscillations in cortical-basal ganglia (BG) circuits have been implicated in normal movement suppression and motor impairment in Parkinson’s disease. To dissect the functional correlates of these rhythms we compared neural activity during four distinct variants of a cued choice task in rats. Brief beta (~20 Hz) oscillations occurred simultaneously throughout the cortical-BG network, both spontaneously and at precise moments of task performance. Beta phase was rapidly reset in response to salient cues, yet increases in beta power were not rigidly linked to cues, movements, or movement suppression. Rather, beta power was enhanced after cues were used to determine motor output. We suggest that beta oscillations reflect a postdecision stabilized state of cortical-BG networks, which normally reduces interference from alternative potential actions. The abnormally strong beta seen in Parkinson’s Disease may reflect overstabilization of these networks, producing pathological persistence of the current motor state.
PMCID: PMC3463873  PMID: 22325204
17.  Comparison of red blood cell survival in sheep determined using red blood cells labeled with either biotin at multiple densities or [14C]cyanate: validation of a model to study human physiology and disease 
Transfusion  2012;52(5):963-973.
Measurement of red blood cell (RBC) survival (RCS) is important for investigating pathophysiology and treatment of anemia. Our objective was to validate the multidensity biotin method for RCS determination in sheep, a commonly used model of RBC physiology. [14C]Cyanate served as the reference method for long-term RCS because the 51Cr method (the reference method for humans) is not reliable in sheep.
Aliquots of autologous RBCs from eight adult sheep were labeled with [14C]cyanate and four separate densities of biotin (BioRBCs) and reinfused. Short-term RCS was assessed by posttransfusion recovery at 24 hours (PTR24); long-term RCS was assessed by the time to 50% survival (T50) and mean potential life span (MPL).
Values for PTR24 of the four BioRBC densities were not different. Values for RCS as reflected by T50 and MPL were nearly identical for [14C]cyanate and the two intermediate-density BioRBC populations. In contrast, the lowest-density BioRBC population survived slightly longer (p < 0.01), but with a difference of no clinical significance. The highest-density BioRBC population importantly shortened RCS (p < 0.01 compared to the two intermediate densities).
This study provides evidence that BioRBCs labeled at four biotin densities can be used to independently and simultaneously measure short-term RCS and that BioRBCs labeled at the three lowest biotin densities can be used to accurately and simultaneously measure long-term RCS. Because the sheep RBC model is comparable to humans, this nonradioactive method has promise for use in RBC kinetic studies in neonates and pregnant women.
PMCID: PMC3564637  PMID: 22229348
18.  Decision support for hospital bed management using adaptable individual length of stay estimations and shared resources 
Elective patient admission and assignment planning is an important task of the strategic and operational management of a hospital and early on became a central topic of clinical operations research. The management of hospital beds is an important subtask. Various approaches have been proposed, involving the computation of efficient assignments with regard to the patients’ condition, the necessity of the treatment, and the patients’ preferences. However, these approaches are mostly based on static, unadaptable estimates of the length of stay and, thus, do not take into account the uncertainty of the patient’s recovery. Furthermore, the effect of aggregated bed capacities have not been investigated in this context. Computer supported bed management, combining an adaptable length of stay estimation with the treatment of shared resources (aggregated bed capacities) has not yet been sufficiently investigated. The aim of our work is: 1) to define a cost function for patient admission taking into account adaptable length of stay estimations and aggregated resources, 2) to define a mathematical program formally modeling the assignment problem and an architecture for decision support, 3) to investigate four algorithmic methodologies addressing the assignment problem and one base-line approach, and 4) to evaluate these methodologies w.r.t. cost outcome, performance, and dismissal ratio.
The expected free ward capacity is calculated based on individual length of stay estimates, introducing Bernoulli distributed random variables for the ward occupation states and approximating the probability densities. The assignment problem is represented as a binary integer program. Four strategies for solving the problem are applied and compared: an exact approach, using the mixed integer programming solver SCIP; and three heuristic strategies, namely the longest expected processing time, the shortest expected processing time, and random choice. A baseline approach serves to compare these optimization strategies with a simple model of the status quo. All the approaches are evaluated by a realistic discrete event simulation: the outcomes are the ratio of successful assignments and dismissals, the computation time, and the model’s cost factors.
A discrete event simulation of 226,000 cases shows a reduction of the dismissal rate compared to the baseline by more than 30 percentage points (from a mean dismissal ratio of 74.7% to 40.06% comparing the status quo with the optimization strategies). Each of the optimization strategies leads to an improved assignment. The exact approach has only a marginal advantage over the heuristic strategies in the model’s cost factors (≤3%). Moreover,this marginal advantage was only achieved at the price of a computational time fifty times that of the heuristic models (an average computing time of 141 s using the exact method, vs. 2.6 s for the heuristic strategy).
In terms of its performance and the quality of its solution, the heuristic strategy RAND is the preferred method for bed assignment in the case of shared resources. Future research is needed to investigate whether an equally marked improvement can be achieved in a large scale clinical application study, ideally one comprising all the departments involved in admission and assignment planning.
PMCID: PMC3621822  PMID: 23289448
19.  Effect of Insulin and an Erythropoietin-Derived Peptide (ARA290) on Established Neuritic Dystrophy and Neuronopathy in Akita (Ins2Akita) Diabetic Mouse Sympathetic Ganglia 
Experimental neurology  2011;232(2):126-135.
The Akita mouse is a robust model of diabetic autonomic neuropathy which develops severe diabetes following beta cell death, which occurs reproducibly at 3-4 weeks of age, and maintains the diabetic state without therapy for as long as 11 additional months. Neuritic dystrophy and neuronopathy involving prevertebral sympathetic superior mesenteric and celiac ganglia begin to develop within the first two months of onset of diabetes and are progressive with time. We have examined the effect of insulin implants resulting in normoglycemia and injections of ARA290, a small erythropoietin peptide which has no effect on glycemic parameters, on the reversal of established neuritic dystrophy and neuronopathy. We have found that 4 weeks of insulin therapy beginning at 2 months of diabetes resulted in normalization of blood glucose, body weight and HbA1c. Insulin therapy successfully reversed established neuritic dystrophy and neuronopathy to control levels. Numbers of sympathetic neurons were not significantly changed in either 3 month diabetic or insulin treated Akita mice. Treatment with ARA290 for 7 weeks beginning at 4 months of diabetes did not result in altered metabolic severity of diabetes as measured by blood glucose, body weight or HbA1c levels. ARA290 treatment was able to decrease neuritic dystrophy by 55-74% compared to untreated diabetics or in comparison to a separate group of diabetic animals representing the 4 month treatment onset point. Surprisingly, there was no effect of ARA290 on ganglionic neuron number or ongoing neuronopathy (pale/degenerating neurons) in diabetic Akita mice during this time period. The development of neuroprotective EPO-like peptides may provide a possible future therapy for this debilitating complication of diabetes; however, it appears that discrete elements may be differentially targeted by the diabetic state and may require selective therapy.
PMCID: PMC3202026  PMID: 21872588
diabetes; Akita mouse; neuritic dystrophy; neuronopathy; erythropoietin; insulin; sympathetic ganglia
20.  Ferromagnetic Nanoscale Electron Correlation Promoted by Organic Spin-Dependent Delocalization 
Journal of the American Chemical Society  2009;131(51):18304-18313.
We describe the electronic structure and the origin of ferromagnetic exchange coupling in two new metal complexes, NN-SQ-CoIII(py)2Cat-NN (1) and NN-Ph-SQ-CoIII(py)2Cat-Ph-NN (2) (NN = nitronylnitroxide radical, Ph = 1,4-phenylene, SQ = S = 1/2 semiquinone radical, Cat = S = 0 catecholate, and py = pyridine). Near-IR electronic absorption spectroscopy for 1 and 2 reveals a low energy optical band that has been assigned as a Ψu → Ψg transition involving bonding and antibonding linear combinations of delocalized dioxolene (SQ/Cat) valence frontier molecular orbitals. The ferromagnetic exchange interaction in 1 is so strong that only the high-spin quartet state (ST = 3/2) is thermally populated at temperatures up to 300 K. The temperature-dependent magnetic susceptibility data for 2 reveals that an excited state spin doublet (ST = 1/2) is populated at higher temperatures, indicating that the phenylene spacer modulates the magnitude of the magnetic exchange. The valence delocalization within the dioxolene dyad of 2 results in ferromagnetic alignment of two localized NN radicals separated by over 22 Å. The ferromagnetic exchange in 1 and 2 results from a spin-dependent delocalization (double exchange type) process and the origin of this strong electron correlation has been understood in terms of a valence bond configuration interaction (VBCI) model. We show that ferromagnetic coupling promoted by organic mixed-valency provides keen insight into the ability of single molecules to communicate spin information over nanoscale distances. Furthermore, the strong interaction between the itinerant dioxolene electron and localized NN electron spins impacts our ability to understand the exchange interaction between delocalized electrons and pinned magnetic impurities in technologically important dilute magnetic semiconductor materials. The long correlation length (22 Å) of the itinerant electron that mediates this coupling indicates that high-spin π-delocalized organic molecules could find applications as nanoscale spin-polarized electron injectors and molecular wires.
PMCID: PMC3505726  PMID: 19928960
21.  Usp14 Deficiency Increases Tau Phosphorylation without Altering Tau Degradation or Causing Tau-Dependent Deficits 
PLoS ONE  2012;7(10):e47884.
Regulated protein degradation by the proteasome plays an essential role in the enhancement and suppression of signaling pathways in the nervous system. Proteasome-associated factors are pivotal in ensuring appropriate protein degradation, and we have previously demonstrated that alterations in one of these factors, the proteasomal deubiquitinating enzyme ubiquitin-specific protease 14 (Usp14), can lead to proteasome dysfunction and neurological disease. Recent studies in cell culture have shown that Usp14 can also stabilize the expression of over-expressed, disease-associated proteins such as tau and ataxin-3. Using Usp14-deficient axJ mice, we investigated if loss of Usp14 results in decreased levels of endogenous tau and ataxin-3 in the nervous system of mice. Although loss of Usp14 did not alter the overall neuronal levels of tau and ataxin-3, we found increased levels of phosphorylated tau that correlated with the onset of axonal varicosities in the Usp14-deficient mice. These changes in tau phosphorylation were accompanied by increased levels of activated phospho-Akt, phosphorylated MAPKs, and inactivated phospho-GSK3β. However, genetic ablation of tau did not alter any of the neurological deficits in the Usp14-deficient mice, demonstrating that increased levels of phosphorylated tau do not necessarily lead to neurological disease. Due to the widespread activation of intracellular signaling pathways induced by the loss of Usp14, a better understanding of the cellular pathways regulated by the proteasome is required before effective proteasomal-based therapies can be used to treat chronic neurological diseases.
PMCID: PMC3483306  PMID: 23144711
22.  Rituximab-Associated Progressive Multifocal Leukoencephalopathy in Rheumatoid Arthritis 
Archives of neurology  2011;68(9):1156-1164.
To describe the development of progressive multifocal leukoencephalopathy (PML) in patients with rheumatoid arthritis (RA) treated with rituximab.
Case study.
Clinical care for patients with rheumatologic diseases. Most were referred to academic centers for care after diagnosis (Washington University, St Louis, Missouri; Karolinska Insitute, Stockholm, Sweden; and Royal Melbourne Hospital, Melbourne, Australia) while one was cared for in a neurology practice in Dallas, Texas, with consultation by an academic neurovirologist from the University of Colorado in Denver.
Four patients developing PML in the setting of rituximab therapy for RA.
Rituximab therapy.
Main Outcome Measures
Clinical and pathological observations.
Four patients from an estimated population of 129 000 exposed to rituximab therapy for RA are reported in whom PML developed after administration of this drug. All were women older than 50 years, commonly with Sjögren syndrome and a history of treatment for joint disease ranging from 3 to 14 years. One case had no prior biologic and minimal immunosuppressive therapy. Progressive multifocal leukoencephalopathy presented as a progressive neurological disorder, with diagnosis confirmed by detection of JC virus DNA in the cerebrospinal fluid or brain biopsy specimen. Two patients died in less than 1 year from PML diagnosis, while 2 remain alive after treatment withdrawal. Magnetic resonance scans and tissue evaluation confirmed the frequent development of inflammatory PML during the course of the disease.
These cases suggest an increased risk, about 1 case per 25 000 individuals, of PML in patients with RA being treated with rituximab. Inflammatory PML may occur in this setting even while CD20 counts remain low.
PMCID: PMC3428054  PMID: 21555606
23.  Cross-frequency phase-phase coupling between theta and gamma oscillations in the hippocampus 
The Journal of Neuroscience  2012;32(2):423-435.
Neuronal oscillations allow for temporal segmentation of neuronal spikes. Interdependent oscillators can integrate multiple layers of information. We examined phase-phase coupling of theta and gamma oscillators in the CA1 region of rat hippocampus during maze exploration and REM sleep. Hippocampal theta waves were asymmetric, and estimation of the spatial position of the animal was improved by identifying the waveform-based phase of spiking, compared to traditional methods used for phase estimation. Using the waveform-based theta phase, 3 distinct gamma bands were identified: slow gammaS (30-50 Hz), mid-frequency gammaM (50-90 Hz) and fast gammaF (90-150 Hz or epsilon band). The amplitude of each sub-band was modulated by the theta phase. In addition, we found reliable phase-phase coupling between theta and both gammaS and gammaM but not gammaF oscillators. We suggest that cross-frequency phase coupling can support multiple time-scale control of neuronal spikes within and across structures.
PMCID: PMC3293373  PMID: 22238079
24.  Red blood cell (RBC) survival determined in humans using RBCs labeled at multiple biotin densities 
Transfusion  2010;51(5):1047-1057.
Safe, accurate methods permitting simultaneous and/or repeated measurement of red blood cell (RBC) survival (RCS) are important to investigate pathophysiology and therapy of anemia. Methods using chromium 51 (51Cr) -labeled RBCs are unacceptable for infants, children, and pregnant women. We report RCS measured in vivo using RBCs labeled with several densities of biotin (BioRBCs).
Aliquots of autologous RBCs from eight healthy adult subjects were labeled separately at four discrete biotin densities, mixed, and infused. The proportion of each population of BioRBCs circulating was determined serially by flow cytometry over 20 weeks. For each population, RCS was assessed by the following: 1) post-transfusion BioRBC recovery at 24 hour (PTR24); 2) time to decrease to 50% of the enrichment at 24 hours (T50); and 3) mean potential lifespan (MPL).
Among the four BioRBC densities, no significant differences in PTR24 were observed. T50 and MPL were similar for the two lowest BioRBC densities. In contrast, the two highest BioRBC densities demonstrated progressively decreased T50 and MPL.
RBCs labeled at four biotin densities can be used to independently and accurately measure PTR24 and two lowest biotin densities can accurately quantitate long-term RCS. This method provides a tool for investigating anemia in infants, fetuses, and pregnant women with the following advantages over the standard 51Cr method: 1) study subjects are not exposed to radiation; 2) small blood volumes (e.g., 20 μL) are required; and 3) multiple independent RCS measurements can be made simultaneously in the same individual.
PMCID: PMC3089718  PMID: 21062290
25.  Radial Diffusivity Predicts Demyelination in ex-vivo Multiple Sclerosis Spinal Cords 
NeuroImage  2011;55(4):1454-1460.
Correlation of diffusion tensor imaging (DTI) with histochemical staining for demyelination and axonal damage in multiple sclerosis (MS) ex vivo human cervical spinal cords.
In MS, demyelination, axonal degeneration, and inflammation contribute to disease pathogenesis to variable degrees. Based upon in vivo animal studies with acute injury and histopathologic correlation, we hypothesized that DTI can differentiate between axonal and myelin pathologies within humans.
DTI was performed at 4.7 Tesla on 9 MS and 5 normal control fixed cervical spinal cord blocks following autopsy. Sections were then stained for Luxol fast blue (LFB), Bielschowsky silver, and hematoxylin and eosin (H&E). Regions of interest (ROIs) were graded semi-quantitatively as normal myelination, mild (<50%) demyelination, or moderate-severe (>50%) demyelination. Corresponding axonal counts were manually determined on Bielschowsky silver. ROIs were mapped to co-registered DTI parameter slices. DTI parameters evaluated included standard quantitative assessments of apparent diffusion coefficient (ADC), relative anisotropy (RA), axial diffusivity and radial diffusivity. Statistical correlations were made between histochemical gradings and DTI parameters using linear mixed models. Results: Within ROIs in MS subjects, increased radial diffusivity distinguished worsening severities of demyelination. Relative anisotropy was decreased in the setting of moderate-severe demyelination compared to normal areas and areas of mild demyelination. Radial diffusivity, ADC, and RA became increasingly altered within quartiles of worsening axonal counts. Axial diffusivity did not correlate with axonal density (p=0.091).
Increased radial diffusivity can serve as a surrogate for demyelination. However, radial diffusivity was also altered with axon injury, suggesting that this measure is not pathologically specific within chronic human MS tissue. We propose that radial diffusivity can serve as a marker of overall tissue integrity within chronic MS lesions. This study provides pathologic foundation for on-going in vivo DTI studies in MS.
PMCID: PMC3062747  PMID: 21238597
Multiple Sclerosis; Diffusion Tensor Imaging; Post mortum; Spinal cord

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