Scale-Free Functional Brain Networks Exhibit Increased Connectivity, Are More Integrated and Less Segregated in Patients with Parkinson’s Disease following Dopaminergic Treatment

Dopaminergic treatment (DT), the standard therapy for Parkinson’s disease (PD), alters the dynamics of functional brain networks at specific time scales. Here, we explore the scale-free functional connectivity (FC) in the PD population and how it is affected by DT. We analyzed the electroencephalogram of: (i) 15 PD patients during DT (ON) and after DT washout (OFF) and (ii) 16 healthy control individuals (HC). We estimated FC using bivariate focus-based multifractal analysis, which evaluated the long-term memory (H(2)) and multifractal strength (ΔH15) of the connections. Subsequent analysis yielded network metrics (node degree, clustering coefficient and path length) based on FC estimated by H(2) or ΔH15. Cognitive performance was assessed by the Mini Mental State Examination (MMSE) and the North American Adult Reading Test (NAART). The node degrees of the ΔH15 networks were significantly higher in ON, compared to OFF and HC, while clustering coefficient and path length significantly decreased. No alterations were observed in the H(2) networks. Significant positive correlations were also found between the metrics of H(2) networks and NAART scores in the HC group. These results demonstrate that DT alters the multifractal coupled dynamics in the brain, warranting the investigation of scale-free FC in clinical and pharmacological studies

Thymic Atrophy and Apoptosis of CD4+CD8+ Thymocytes in the Cuprizone Model of Multiple Sclerosis.

Previous studies on the degenerative animal model of multiple sclerosis suggested that the copper-chelator cuprizone might directly suppress T-cell functions. Peripheral T-cell function in the cuprizone model has already been explored; therefore, in the present study, we investigated, for the first time, how cuprizone feeding affects the thymus, the organ of T-cell maturation and selection. We found that even one week of cuprizone treatment induced significant thymic atrophy, affecting the cortex over the medulla. Fluorescent microscopy and flow-cytometric analyses of thymi from cuprizone- and vehicle-treated mice indicated that eradication of the cluster of the differentiation-4 (CD4)-CD8 double-positive T-cell subset was behind the substantial cell loss. This result was confirmed with CD3-CD4-CD8 triple-staining experiments. Ultrastructurally, we observed degraded as well as enlarged mitochondria, myelin-bodies, large lipid droplets, and large lysosomes in the thymi of cuprizone-treated mice. Some of these features were similar to those in physiological and steroid-induced accelerated aging. According to our results, apoptosis was mainly of mitochondrial origin mediated by both caspase-3- and apoptosis inducing factor-mediated mechanisms. Additionally, mitogen activated protein kinase activation and increased pro-apoptotic B cell lymphoma-2 family protein expression were the major underlying processes. Our results do not indicate a functional relationship between cuprizone-induced thymus involution and the absence of inflammatory responses or the selective demyelination observed in the cuprizone model. On the other hand, due to the reversible nature of cuprizone's deleterious effects, the cuprizone model could be valuable in studying thymus regeneration as well as remyelination processes

Effect of sildenafil on Akt activation.

<p>Effect of Sildenafil and MCT treatment on Akt expression (A) and activation (B) as well as on GSK-3β expression (C) and phosphorylation (D) was determined by immunoblotting utilizing protein- and phosphorylation specific primary antibodies. The bar diagrams represent pixel volumes±S.E.M. of pAkt and pGSK-3β bands. The bands were normalized to the appropriate Akt and GSK-3β bands. *** significantly different from Sham group (p≤0.05), +++ significantly different from PH group (p≤0.05) by ANOVA, post hoc Bonferroni test. Phospho-Akt and GSK-3β are denoted as pAkt and pGSK-3β.</p

Effects of sildenafil treatment on cytokine expressions.

<p>Nitrocellulose based rat cytokine array assay was performed on lung homogenates of untreated (Sham), sildenagfil (Sham+SLD), MCT (PH) and MCT plus sildenafil treated (PH+SLD) rats (n = 3–4). Bar diagrams represent mean±S.E.M. pixel densities of the immunoblots. *** significantly different from Sham group (p≤0.05), +++ significantly different from PH group (p≤0.05) by ANOVA, post hoc Bonferroni test.</p

Effects of sildenafil treatment on NFκB activation and nuclear translocation.

<p>NFκB activation and nuclear translocation was assessed by immunoblotting (A) and immunohistochemistry (B) utilizing phospho-NF-κB p65 (A,B) and NF-κB (A) specific primary antibodies. Bar diagrams (A) of the mean pixel densities±S.E.M. and representative immunohistochemistry images (B) of 3 to 4 animals are presented. The phospho-NFκB p65 bands were normalized to the NFκB band. *** significantly different from Sham group (p≤0.05), +++ significantly different from PH group (p≤0.05) by ANOVA, post hoc Bonferroni test.</p

Effect of sildenafil on ERK1/2 and p38 MAPK activation.

<p>Effect of Sildenafil and MCT treatment on ERK 1 and 2 expression (A) and activation (B) as well as on p38 MAPK expression (C) and phosphorylation (D) was determined by immunoblotting utilizing protein- and phosphorylation specific primary antibodies. The bar diagrams represent pixel volumes±S.E.M. of pERK1/2 and pp38 MAPK bands. The bands were normalized to the appropriate ERK1/2 and p38 MAPK bands. *** significantly different from Sham group (p≤0.05), +++ significantly different from PH group (p≤0.05) by ANOVA, post hoc Bonferroni test. Phospho-ERK1/2 and p38 MAPK are denoted as pERK1/2 and pp38 MAPK.</p

Evaluation of Dexterity in Insulin-Treated Patients with Type 1 and Type 2 Diabetes Mellitus

BACKGROUND: Daily routine for insulin-treated patients with diabetes mellitus requires correct performance of self-monitoring of blood glucose and insulin injections several times a day. Dexterity skills may play an important role in the performance efficacy of these procedures. METHODS: We collected data of insulin-treated (>10 years) patients with different age ranges [healthy controls, 14 female/11 male, age (mean ± standard deviation) 55 ± 7 years; type 1 diabetes mellitus (T1DM) patients, 12/13, 45 ± 9 years, disease duration 23.9 ± 6.5 years; T2DM patients, 8/17, 64 ± 6 years, 16.2 ± 6.9 years; T2DM patients (>70 years of age), 9/16, 75 ± 4 years, 19.7 ± 7.0 years]. After assessment of neuropathy (temperature, pain, and vibration perception), the patients participated in two dexterity test batteries [Jebsen–Taylor hand-function test (JHFT) and motoric performance series (MPS)]. RESULTS: Patients with type 2 diabetes showed disturbed vibration perception as compared to the other groups. The dexterity results were influenced by age to a large extent. Older T2DM patients performed worst in the majority of the subtests (e.g., JHFT, writing nondominant hand: control, 40.8 ± 11.7 s; T1DM, 46.3 ± 50.9 s, not significant versus control; old T2DM, 68.1 ± 29.5 s, p < .05; young T2DM, 52.5 ± 26.2 s, p < .05). Patients with type 1 diabetes showed similar JHFT and MPS results than the 10-year-older control subjects and performed outside of the age-dependent normal reference range. CONCLUSIONS: Manual skills and dexterity differed between the groups, and age-corrected reduced skills were common in both T1DM and T2DM patients in this study. Our findings underline the importance of considering dexterity and manual skills when designing medical devices for patients with diabetes mellitus

Effect of cuprizone on thymic epithelial cells.

<p>Four week-old male mice were treated with cuprizone for one week, then immune-staining (A) with FITC-labelled anti-EpCAM1 (green) and PE-labelled anti-Ly-51 (red) antibodies was performed on thymic sections of untreated (Control) and cuprizone-treated (CPZ) mice. Representative images (A) are presented of the green channel (top panels), the red channel (middle panels) and the merged channels (bottom panels) of three independent experiments, including at least three animals in each group for each experiment. Fluorescent photographs were taken using a 10x objective. The scale bar indicates 200 μm. In a parallel experiment, thymic MHC II and AIRE mRNA expression (B) was determined by using qPCR analysis in untreated (grey bars) and cuprizone-treated (black bars) mice. Results are presented as fold change, mean + SEM (n≥9). Significant difference from control; *p<0.05.</p

Macroscopic changes of the thymi upon cuprizone treatment.

<p>Four week-old male mice were treated with cuprizone for one week. Representative photographs (A) of the open chest of a control (Cont.) and a cuprizone-treated (CPZ) animal are demonstrated. Arrows point to the thymi of the mice. The scale bar indicates 5 mm. Thymus mass (B), body mass (C) and relative thymus mass (thymus tissue mass/ body mass) (E) of control (grey bars) and cuprizone-treated (black bars) animals are presented as bar diagrams, mean + SEM (n≥9). * denotes a significant difference from control p<0.05.</p

Effect of cuprizone treatment on death pathway and signalling proteins in the thymus.

<p>Four week-old male mice were treated with cuprizone for three days. Steady-state cytoplasmic levels of cytochrome C (Cyt C) (A), caspase 3 (casp 3) (A), cleaved caspase 3 (cleav. casp 3) (A) and nuclear apoptosis inducing factor (AIF) content (A), as well as cellular levels of Bad (B), BimEL (B), BimL (B) and Bax (B) were assessed in the thymi of untreated (Cont., grey bars) and cuprizone-treated (CPZ, black bars) mice by using specific primary antibodies and immunoblotting. The activation state of Bad (p-Bad) (B), JNK (p-JNK) (C), ERK (p-ERK) (C) and p38 MAPK (p-p38) (C) was also determined by using phosphorylation-specific primary antibodies and immunoblotting. GAPDH (A-C) and histone H1 (His H1) (A) were used as loading controls for cytoplasmic/cellular and nuclear fractions, respectively. Results are presented as representative immunoblots and bar diagrams, mean + SEM (n≥9). Significant difference from control; *p<0.05. Please note that the y-axis in B is broken to accommodate the very high Bax value.</p