Reduced Responsiveness to Long-Term Monocular Deprivation of Parvalbumin Neurons Assessed by c-Fos Staining in Rat Visual Cortex

Background

It is generally assumed that visual cortical cells homogeneously shift their ocular dominance (OD) in response to monocular deprivation (MD), however little experimental evidence directly supports this notion. By using immunohistochemistry for the activity-dependent markers c-Fos and Arc, coupled with staining for markers of inhibitory cortical sub-populations, we studied whether long-term MD initiated at P21 differentially affects visual response of inhibitory neurons in rat binocular primary visual cortex.

Methodology/Principal Findings

The inhibitory markers GAD67, parvalbumin (PV), calbindin (CB) and calretinin (CR) were used. Visually activated Arc did not colocalize with PV and was discarded from further studies. MD decreased visually induced c-Fos activation in GAD67 and CR positive neurons. The CB population responded to MD with a decrease of CB expression, while PV cells did not show any effect of MD on c-Fos expression. The persistence of c-Fos expression induced by deprived eye stimulation in PV cells is not likely to be due to a particularly low threshold for activity-dependent c-Fos induction. Indeed, c-Fos induction by increasing concentrations of the GABAA antagonist picrotoxin in visual cortical slices was similar between PV cells and the other cortical neurons.

Conclusion

These data indicate that PV cells are particularly refractory to MD, suggesting that different cortical subpopulation may show different response to MD.     

Immunolocalization of the Tumor-Sensitive Calmodulin-Like Protein CALML3 in Normal Human Skin and Hyperproliferative Skin Disorders

Background and Objective

Calmodulin-like protein CALML3 is an epithelial-specific protein regulated during keratinocyte differentiation in vitro. CALML3 expression is downregulated in breast cancers and transformed cell lines making it an attractive marker for tumor formation. The objective of this study was to survey CALML3 localization in normal epidermis and in hyperproliferative skin diseases including actinic keratosis, squamous and basal cell carcinoma as well as verruca and psoriasis and to compare CALML3 immunoreactivity with the proliferation marker Ki-67.

Methods

Paraffin-embedded tissue sections from normal human skin and hyperproliferative skin disorders were examined by immunohistochemistry and analyzed for localization and expression of CALML3 and Ki-67.

Results

CALML3 was strongly expressed in differentiating layers of normal skin, staining the periphery in suprabasal cells and exhibiting nuclear localization in the stratum granulosum. CALML3 nuclear localization was inversely correlated to Ki-67 staining in each disease, indicating that CALML3 nuclear presence is related to terminal cell differentiation and postmitotic state.

Conclusions

Increased CALML3 expression in suprabasal layers is characteristic for differentiating keratinocytes in normal epidermis, and nuclear expression of CALML3 inversely correlates with expression of the proliferation marker Ki-67. This suggests that CALML3 is a useful marker for normal and benign hyperplastic epidermal development, whereas the loss of nuclear CALML3 indicates progression to a proliferative and potentially malignant phenotype.     

An analysis of the cross-reactivity of autoantibodies to GAD65 and GAD67 in diabetes

Background

Autoantibodies to GAD65 (anti-GAD65) are present in the sera of 70–80% of patients with type 1 diabetes (T1D), but antibodies to the structurally similar 67 kDa isoform GAD67 are rare. Antibodies to GAD67 may represent a cross-reactive population of anti-GAD65, but this has not been formally tested.

Methodology/Principal Findings

In this study we examined the frequency, levels and affinity of anti-GAD67 in diabetes sera that contained anti-GAD65, and compared the specificity of GAD65 and GAD67 reactivity. Anti-GAD65 and anti-GAD67 were measured by radioimmunoprecipitation (RIP) using ¹²⁵I labeled recombinant GAD65 and GAD67. For each antibody population, the specificity of the binding was measured by incubation with 100-fold excess of unlabeled GAD in homologous and heterologous inhibition assays, and the affinity of binding with GAD65 and GAD67 was measured in selected sera. Sera were also tested for reactivity to GAD65 and GAD67 by immunoblotting. Of the 85 sera that contained antibodies to GAD65, 28 contained anti–GAD67 measured by RIP. Inhibition with unlabeled GAD65 substantially or completely reduced antibody reactivity with both ¹²⁵I GAD65 and with ¹²⁵I GAD67. In contrast, unlabeled GAD67 reduced autoantibody reactivity with ¹²⁵I GAD67 but not with ¹²⁵I GAD65. Both populations of antibodies were of high affinity (>10¹⁰ l/mol).

Conclusions

Our findings show that autoantibodies to GAD67 represent a minor population of anti-GAD65 that are reactive with a cross-reactive epitope found also on GAD67. Experimental results confirm that GAD65 is the major autoantigen in T1D, and that GAD67 per se has very low immunogenicity. We discuss our findings in light of the known similarities between the structures of the GAD isoforms, in particular the location of a minor cross-reactive epitope that could be induced by epitope spreading.     

Differentiation of mucinous from non-mucinous pancreatic cyst fluid using dual-stained, 1 dimensional polyacrylamide gel electrophoresis

Background

Pancreatic cysts are being increasingly identified in patients. Mucinous cysts have malignant potential whereas non-mucinous cysts do not. Distinguishing potentially malignant cysts from harmless ones by the characterization of cyst fluid contents remains a difficult problem. This study was undertaken to determine whether cyst fluid mucin glycoprotein analysis could differentiate mucinous from non-mucinous pancreatic cysts.

Methods

Cyst fluid from 28 patients who underwent resection of a pancreatic cyst was used for the study. In each case the type of cyst was histologically identified. One dimensional SDS polyacrylamide gel electrophoresis (1D-SDS PAGE) was performed on cyst fluid samples. For the detection of the separated proteins, we employed a novel dual staining technique. The gel was first stained with periodic acid Schiff (PAS), a mucin histochemical stain followed by a secondary protein staining with Simply Blue Safestain (Invitrogen).

Results

Visual scoring (based on the presence of mucins) gave a sensitivity of 95%, a specificity of 100%, a positive predictive value of 100%, and a negative predictive value of 88% for prediction of mucinous histology.

Conclusions

One dimensional SDS polyacrylamide gel electrophoresis of pancreatic cyst fluid, followed by mucin (PAS) and protein (Simply Blue Safestain) staining, provides a means of concentrating and visualizing mucins, which allows the accurate differentiation of mucinous from non-mucinous histology in pancreatic cysts.     

Development of IGF Signaling Antibody Arrays for the Identification of Hepatocellular Carcinoma Biomarkers

Purpose

Our objective was to develop a system to simultaneously and quantitatively measure the expression levels of the insulin-like growth factor (IGF) family proteins in numerous samples and to apply this approach to profile the IGF family proteins levels in cancer and adjacent tissues from patients with hepatocellular carcinoma (HCC).

Experimental Design

Antibodies against ten IGF family proteins (IGF-1, IGF-1R, IGF-2, IGF-2R, IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-6, and Insulin) were immobilized on the surface of a glass slide in an array format to create an IGF signaling antibody array. Tissue lysates prepared from patient''s liver cancer tissues and adjacent tissues were then applied to the arrays. The proteins captured by antibodies on the arrays were then incubated with a cocktail of biotinylated detection antibodies and visualized with a fluorescence detection system. By comparison with standard protein amount, the exact protein concentrations in the samples can be determined. The expression levels of the ten IGF family proteins in 25 pairs of HCC and adjacent tissues were quantitatively measured using this novel antibody array technology. The differential expression levels between cancer tissues and adjacent tissues were statistically analyzed.

Results

A novel IGF signaling antibody array was developed which allows the researcher to simultaneously detect ten proteins involved in IGF signal pathway with high sensitivity and specificity. Using this approach, we found that the levels of IGF-2R and IGFBP-2 in HCC tissues were higher than those in adjacent tissues.

Conclusion

Our IGF signaling antibody array which can detect the expression of ten IGF family members with high sensitivity and specificity will undoubtedly prove a powerful tool for drug and biomarker discovery.     

7 Tesla Magnetic Resonance Imaging to Detect Cortical Pathology in Multiple Sclerosis

Background

Neocortical lesions (NLs) are an important pathological component of multiple sclerosis (MS), but their visualization by magnetic resonance imaging (MRI) remains challenging.

Objectives

We aimed at assessing the sensitivity of multi echo gradient echo (ME-GRE) T₂ ^*-weighted MRI at 7.0 Tesla in depicting NLs compared to myelin and iron staining.

Methods

Samples from two MS patients were imaged post mortem using a whole body 7T MRI scanner with a 24-channel receive-only array. Isotropic 200 micron resolution images with varying T₂ ^* weighting were reconstructed from the ME-GRE data and converted into R₂ ^* maps. Immunohistochemical staining for myelin (proteolipid protein, PLP) and diaminobenzidine-enhanced Turnbull blue staining for iron were performed.

Results

Prospective and retrospective sensitivities of MRI for the detection of NLs were 48% and 67% respectively. We observed MRI maps detecting only a small portion of 20 subpial NLs extending over large cortical areas on PLP stainings. No MRI signal changes suggestive of iron accumulation in NLs were observed. Conversely, R₂ ^* maps indicated iron loss in NLs, which was confirmed by histological quantification.

Conclusions

High-resolution post mortem imaging using R₂ ^* and magnitude maps permits detection of focal NLs. However, disclosing extensive subpial demyelination with MRI remains challenging.     

EGFR protein overexpression correlates with chromosome 7 polysomy and poor prognostic parameters in clear cell renal cell carcinoma

Background

The role of epidermal growth factor (EGF) and its receptor (EGFR) in the pathogenesis and progression of various malignant tumors has long been known, but there is still disagreement concerning prognostic significance of EGFR expression in clear cell renal cell carcinoma (CCRCC). The present study was designed to analyze more objectively the protein EGFR expression in CCRCC and to compare its value with EGFR gene copy number changes and clinicopathologic characteristics including patient survival.

Methods

The protein EGFR expression was analyzed immunohistochemically on 94 CCRCC, and gene copy number alterations of EGFR by FISH analysis on 41 CCRCC selected according to distinct membrane EGFR staining.

Results

Membrane EGFR expression in tumor cells was heterogeneous with respect to the proportion of positive cells and staining intensity. FISH analysis did not reveal EGFR gene amplification, while polysomy of chromosome 7 found in 41% was associated with higher EGFR membrane expression. Moreover, EGFR overexpression was associated with a higher nuclear grade, larger tumor size and shorter patient''s survival, while there was no connection with pathological stage.

Conclusion

In conclusion, the protein expression of EGFR had an impact on prognosis in patients with CCRCC, while an increased copy number of chromosome 7 could be the possible reason for EGFR protein overexpression in the absence of gene amplification.     

Comparison of methods for detection of Blastocystis infection in routinely submitted stool samples, and also in IBS/IBD Patients in Ankara, Turkey

Background

This study compared diagnostic methods for identifying Blastocystis in stool samples, and evaluated the frequency of detection of Blastocystis in patients with irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD).

Results and Discussion

From a set of 105 stool specimens submitted for routine parasitological analysis, 30 were identified as positive for Blastocystis by the culture method. From that group of 30 positives, Lugol''s stain, trichrome staining, and an immunofluorescence assay identified 11, 15, and 26 samples as positive respectively. Using culture as a standard, the sensitivity of Lugol''s stain was 36.7%, trichrome staining was 50%, and the IFA stain was 86.7%. The specificity of Lugol''s stain was 91%, trichrome staining was 100%, and the IFA stain was 97.3%. In the group of 27 IBS and IBD patients, using all methods combined, we detected Blastocystis in 67% (18/27) of the patients. Blastocystis was detected in 33% (2/6) of IBD patients and 76% (16/21) of IBS patients. For comparison, trichrome staining alone, the method most frequently used in many countries, would have only identified Blastocystis infection in 29% (6/21) of the IBS patients. No parasitic co-infections were identified in the IBS/IBD patients. Most Blastocystis-positive IBS/IBD patients were over 36 with an average length of illness of 4.9 years.

Conclusions

Most IBS patients in this study were infected with Blastocystis. IFA staining may be a useful alternative to stool culture, especially if stool specimens have been chemically preserved.     

Cyclical and patch-like GDNF distribution along the basal surface of Sertoli cells in mouse and hamster testes

Background and Aims

In mammalian spermatogenesis, glial cell line-derived neurotrophic factor (GDNF) is one of the major Sertoli cell-derived factors which regulates the maintenance of undifferentiated spermatogonia including spermatogonial stem cells (SSCs) through GDNF family receptor α1 (GFRα1). It remains unclear as to when, where and how GDNF molecules are produced and exposed to the GFRα1-positive spermatogonia in vivo.

Methodology and Principal Findings

Here we show the cyclical and patch-like distribution of immunoreactive GDNF-positive signals and their close co-localization with a subpopulation of GFRα1-positive spermatogonia along the basal surface of Sertoli cells in mice and hamsters. Anti-GDNF section immunostaining revealed that GDNF-positive signals are mainly cytoplasmic and observed specifically in the Sertoli cells in a species-specific as well as a seminiferous cycle- and spermatogenic activity-dependent manner. In contrast to the ubiquitous GDNF signals in mouse testes, high levels of its signals were cyclically observed in hamster testes prior to spermiation. Whole-mount anti-GDNF staining of the seminiferous tubules successfully visualized the cyclical and patch-like extracellular distribution of GDNF-positive granular deposits along the basal surface of Sertoli cells in both species. Double-staining of GDNF and GFRα1 demonstrated the close co-localization of GDNF deposits and a subpopulation of GFRα1-positive spermatogonia. In both species, GFRα1-positive cells showed a slender bipolar shape as well as a tendency for increased cell numbers in the GDNF-enriched area, as compared with those in the GDNF-low/negative area of the seminiferous tubules.

Conclusion/Significance

Our data provide direct evidence of regionally defined patch-like GDNF-positive signal site in which GFRα1-positive spermatogonia possibly interact with GDNF in the basal compartment of the seminiferous tubules.     

The Inhibition of Subchondral Bone Lesions Significantly Reversed the Weight-Bearing Deficit and the Overexpression of CGRP in DRG Neurons,GFAP and Iba-1 in the Spinal Dorsal Horn in the Monosodium Iodoacetate Induced Model of Osteoarthritis Pain

Background

Chronic pain is the most prominent and disabling symptom of osteoarthritis (OA). Clinical data suggest that subchondral bone lesions contribute to the occurrence of joint pain. The present study investigated the effect of the inhibition of subchondral bone lesions on joint pain.

Methods

Osteoarthritic pain was induced by an injection of monosodium iodoacetate (MIA) into the rat knee joint. Zoledronic acid (ZOL), a third generation of bisphosphonate, was used to inhibit subchondral bone lesions. Joint histomorphology was evaluated using X-ray micro computed tomography scanning and hematoxylin-eosin staining. The activity of osteoclast in subchondral bone was evaluated using tartrate-resistant acid phosphatase staining. Joint pain was evaluated using weight-bearing asymmetry, the expression of calcitonin gene-related peptide (CGRP) in the dorsal root ganglion (DRG), and spinal glial activation status using glial fibrillary acidic protein (GFAP) and ionized calcium binding adaptor molecule-1 (Iba-1) immunofluorescence. Afferent neurons in the DRGs that innervated the joints were identified using retrograde fluorogold labeling.

Results

MIA injections induced significant histomorphological alterations and joint pain. The inhibition of subchondral bone lesions by ZOL significantly reduced the MIA-induced weight-bearing deficit and overexpression of CGRP in DRG neurons, GFAP and Iba-1 in the spinal dorsal horn at 3 and 6 weeks after MIA injection; however, joint swelling and synovial reaction were unaffected.

Conclusions

The inhibition of subchondral bone lesions alleviated joint pain. Subchondral bone lesions should be a key target in the management of osteoarthritic joint pain.     

Effects of antenatal glucocorticoid therapy on hippocampal histology of preterm infants

Objective

To investigate if antenatal glucocorticoid treatment has an effect on hippocampal histology of the human preterm newborn.

Patients and Methods

Included were consecutive neonates with a gestational age between 24 and 32 weeks, who were born between 1991 to 2009, who had died within 4 days after delivery and underwent brain autopsy. Excluded were neonates with congenital malformations and neonates treated postnatally with glucocorticoids.The brains were routinely fixed, samples of the hippocampus were stained with haematoxylin and eosin and sections were examined for presence or absence of large and small neurons in regions of the hippocampus. Additional staining with GFAP, neurofilament and vimentin was performed to evaluate gliosis and myelination. The proliferation marker Ki67 was used to evaluate neuronal proliferation. Staining with acid fuchsin-thionin was performed to evaluate ischemic damage.

Results

The hippocampi of ten neonates who had been treated with antenatal glucocorticoids showed a lower density of large neurons (p = 0.01) and neurons irrespective of size (p = 0.02) as compared to eleven neonates who had not been treated with glucocorticoids. No difference was found in density of small neurons, in myelination, gliosis, proliferation or ischemic damage.

Conclusion

We found a significantly lower density of neurons in the hippocampus of neonates after antenatal glucocorticoid treatment. Although the pathophysiological and clinical interpretations of these findings are not clear, they are consistent with those from experiments in mice and rhesus monkeys.     

CFTR expression analysis in human nasal epithelial cells by flow cytometry

Rationale

Unbiased approaches that study aberrant protein expression in primary airway epithelial cells at single cell level may profoundly improve diagnosis and understanding of airway diseases. We here present a flow cytometric procedure to study CFTR expression in human primary nasal epithelial cells from patients with Cystic Fibrosis (CF). Our novel approach may be important in monitoring of therapeutic responses, and better understanding of CF disease at the molecular level.

Objectives

Validation of a panel of CFTR-directed monoclonal antibodies for flow cytometry and CFTR expression analysis in nasal epithelial cells from healthy controls and CF patients.

Methods

We analyzed CFTR expression in primary nasal epithelial cells at single cell level using flow cytometry. Nasal cells were stained for pan-Cytokeratin, E cadherin, and CD45 (to discriminate epithelial cells and leukocytes) in combination with intracellular staining of CFTR. Healthy individuals and CF patients were compared.

Measurements and Main Results

We observed various cellular populations present in nasal brushings that expressed CFTR protein at different levels. Our data indicated that CF patients homozygous for F508del express varying levels of CFTR protein in nasal epithelial cells, although at a lower level than healthy controls.

Conclusion

CFTR protein is expressed in CF patients harboring F508del mutations but at lower levels than in healthy controls. Multicolor flow cytometry of nasal cells is a relatively simple procedure to analyze the composition of cellular subpopulations and protein expression at single cell level.     

Childhood Maltreatment Is Associated with Larger Left Thalamic Gray Matter Volume in Adolescents with Generalized Anxiety Disorder

Background

Generalized anxiety disorder (GAD) is a common anxiety disorder that usually begins in adolescence. Childhood maltreatment is highly prevalent and increases the possibility for developing a variety of mental disorders including anxiety disorders. An earlier age at onset of GAD is significantly related to maltreatment in childhood. Exploring the underpinnings of the relationship between childhood maltreatment and adolescent onset GAD would be helpful in identifying the potential risk markers of this condition.

Methods

Twenty-six adolescents with GAD and 25 healthy controls participated in this study. A childhood trauma questionnaire (CTQ) was introduced to assess childhood maltreatment. All subjects underwent high-resolution structural magnetic resonance scans. Voxel-based morphometry (VBM) was used to investigate gray matter alterations.

Results

Significantly larger gray matter volumes of the right putamen were observed in GAD patients compared to healthy controls. In addition, a significant diagnosis-by-maltreatment interaction effect for the left thalamic gray matter volume was revealed, as shown by larger volumes of the left thalamic gray matter in GAD patients with childhood maltreatment compared with GAD patients without childhood maltreatment as well as with healthy controls with/without childhood maltreatment. A significant positive association between childhood maltreatment and left thalamic gray matter volume was only seen in GAD patients.

Conclusions

These findings revealed an increased volume in the subcortical regions in adolescent GAD, and the alterations in the left thalamus might be involved in the association between childhood maltreatment and the occurrence of GAD.     

β2-Agonists Inhibit TNF-α-Induced ICAM-1 Expression in Human Airway Parasympathetic Neurons

Background

Major basic protein released from eosinophils to airway parasympathetic nerves blocks inhibitory M₂ muscarinic receptors on the parasympathetic nerves, increasing acetylcholine release and potentiating reflex bronchoconstriction. Recruitment of eosinophils to airway parasympathetic neurons requires neural expression of both intercellular adhesion molecular-1 (ICAM-1) and eotaxin. We have shown that inflammatory cytokines induce eotaxin and ICAM-1 expression in parasympathetic neurons.

Objective

To test whether the β₂ agonist albuterol, which is used to treat asthma, changes TNF-alpha-induced eotaxin and ICAM-1 expression in human parasympathetic neurons.

Methods

Parasympathetic neurons were isolated from human tracheas and grown in serum-free medium for one week. Cells were incubated with either (R)-albuterol (the active isomer), (S)-albuterol (the inactive isomer) or (R,S)-albuterol for 90 minutes before adding 2 ng/ml TNF-alpha for another 4 hours (for mRNA) or 24 hours (for protein).

Results and Conclusions

Baseline expression of eotaxin and ICAM-1 were not changed by any isomer of albuterol as measured by real time RT-PCR. TNF-alpha induced ICAM-1 expression was significantly inhibited by (R)-albuterol in a dose dependent manner, but not by (S) or (R,S)-albuterol. Eotaxin expression was not changed by TNF-alpha or by any isomer of albuterol. The β-receptor antagonist propranolol blocked the inhibitory effect of (R)-albuterol on TNF-alpha-induced ICAM-1 expression.

Clinical Implication

The suppressive effect of (R)-albuterol on neural ICAM-1 expression may be an additional mechanism for decreasing bronchoconstriction, since it would decrease eosinophil recruitment to the airway nerves.     

Synaptotagmin-2 is a reliable marker for parvalbumin positive inhibitory boutons in the mouse visual cortex

Background

Inhibitory innervation by parvalbumin (PV) expressing interneurons has been implicated in the onset of the sensitive period of visual plasticity. Immunohistochemical analysis of the development and plasticity of these inhibitory inputs is difficult because PV expression is low in young animals and strongly influenced by neuronal activity. Moreover, the synaptic boutons that PV neurons form onto each other cannot be distinguished from the innervated cell bodies by immunostaining for this protein because it is present throughout the cells. These problems call for the availability of a synaptic, activity-independent marker for PV+ inhibitory boutons that is expressed before sensitive period onset. We investigated whether synaptotagmin-2 (Syt2) fulfills these properties in the visual cortex. Syt2 is a synaptic vesicle protein involved in fast Ca²⁺ dependent neurotransmitter release. Its mRNA expression follows a pattern similar to that of PV throughout the brain and is present in 30–40% of hippocampal PV expressing basket cells. Up to now, no quantitative analyses of Syt2 expression in the visual cortex have been carried out.

Methodology/Principal Findings

We used immunohistochemistry to analyze colocalization of Syt2 with multiple interneuron markers including vesicular GABA transporter VGAT, calbindin, calretinin, somatostatin and PV in the primary visual cortex of mice during development and after dark-rearing.

Conclusions/Significance

We show that in the adult visual cortex Syt2 is only found in inhibitory, VGAT positive boutons. Practically all Syt2 positive boutons also contain PV and vice versa. During development, Syt2 expression can be detected in synaptic boutons prior to PV and in contrast to PV expression, Syt2 is not down-regulated by dark-rearing. These properties of Syt2 make it an excellent marker for analyzing the development and plasticity of perisomatic inhibitory innervations onto both excitatory and inhibitory neurons in the visual cortex.     

The Risk of Cancer in Patients with Generalized Anxiety Disorder: A Nationwide Population-Based Study

Objective

To evaluate the risk of cancer among patients with generalized anxiety disorder (GAD) in a nationwide population-based dataset.

Methods

We recruited newly-diagnosed GAD patients aged 20 years or older without antecedent cancer from the Taiwan National Health Insurance Research database between 2000–2010. Standardized incidence ratios (SIRs) of cancers were calculated in GAD patients, and the subgroup of GAD patients diagnosed by psychiatric specialists.

Results

A total of 559 cancers developed among 19,793 GAD patients with a follow-up of 89,485 person-years (median follow-up of 4.34 years), leading to a significantly increased SIR of 1.14 [95% confidence interval (CI) 1.05–1.24]. Male GAD patients had a significantly increased SIR overall (1.30, 95% CI 1.15–1.46) and for lung and prostate cancer (1.77, 95% CI 1.33–2.30 and 2.17, 95% CI 1.56–2.93, respectively). Patients over 80 years of age also had a significantly increased SIR (1.56, 95% CI 1.25–1.92), especially in males. However, psychiatrist-diagnosed GAD patients did not show increased cancer risk relative to the general population, perhaps due to having fewer physical comorbidities than non-psychiatrist-diagnosed GAD patients.

Conclusion

This study found that overall cancer risk is elevated among patients with GAD. The risk of lung and prostate cancer also increased in male patients with GAD. This increased cancer risk may be due to physical comorbidities and surveillance bias. Further prospective study is necessary to confirm these findings.     

Hyperglycemia induces oxidative stress and impairs axonal transport rates in mice

Background

While hyperglycemia-induced oxidative stress damages peripheral neurons, technical limitations have, in part, prevented in vivo studies to determine the effect of hyperglycemia on the neurons in the central nervous system (CNS). While olfactory dysfunction is indicated in diabetes, the effect of hyperglycemia on olfactory receptor neurons (ORNs) remains unknown. In this study, we utilized manganese enhanced MRI (MEMRI) to assess the impact of hyperglycemia on axonal transport rates in ORNs. We hypothesize that (i) hyperglycemia induces oxidative stress and is associated with reduced axonal transport rates in the ORNs and (ii) hyperglycemia-induced oxidative stress activates the p38 MAPK pathway in association with phosphorylation of tau protein leading to the axonal transport deficits.

Research Design and Methods

T₁-weighted MEMRI imaging was used to determine axonal transport rates post-streptozotocin injection in wildtype (WT) and superoxide dismutase 2 (SOD2) overexpressing C57Bl/6 mice. SOD2 overexpression reduces mitochondrial superoxide load. Dihydroethidium staining was used to quantify the reactive oxygen species (ROS), specifically, superoxide (SO). Protein and gene expression levels were determined using western blotting and Q-PCR analysis, respectively.

Results

STZ-treated WT mice exhibited significantly reduced axonal transport rates and significantly higher levels of ROS, phosphorylated p38 MAPK and tau protein as compared to the WT vehicle treated controls and STZ-treated SOD2 mice. The gene expression levels of p38 MAPK and tau remained unchanged.

Conclusion

Increased oxidative stress in STZ-treated WT hyperglycemic mice activates the p38 MAPK pathway in association with phosphorylation of tau and attenuates axonal transport rates in the olfactory system. In STZ-treated SOD-overexpressing hyperglycemic mice in which superoxide levels are reduced, these deficits are reversed.     

Induction of cell stress in neurons from transgenic mice expressing yellow fluorescent protein: implications for neurodegeneration research

Background

Mice expressing fluorescent proteins in neurons are one of the most powerful tools in modern neuroscience research and are increasingly being used for in vivo studies of neurodegeneration. However, these mice are often used under the assumption that the fluorescent proteins present are biologically inert.

Methodology/Principal Findings

Here, we show that thy1-driven expression of yellow fluorescent protein (YFP) in neurons triggers multiple cell stress responses at both the mRNA and protein levels in vivo. The presence of YFP in neurons also subtly altered neuronal morphology and modified the time-course of dying-back neurodegeneration in experimental axonopathy, but not in Wallerian degeneration triggered by nerve injury.

Conclusions/Significance

We conclude that fluorescent protein expressed in thy1-YFP mice is not biologically inert, modifies molecular and cellular characteristics of neurons in vivo, and has diverse and unpredictable effects on neurodegeneration pathways.