Regulatory Action of Plasma from Patients with Obesity and Diabetes towards Muscle Cells Differentiation and Bioenergetics Revealed by the C2C12 Cell Model and MicroRNA Analysis.

Obesity and type 2 diabetes mellitus (T2DM) are often combined and pathologically affect many tissues due to changes in circulating bioactive molecules. In this work, we evaluated the effect of blood plasma from obese (OB) patients or from obese patients comorbid with diabetes (OBD) on skeletal muscle function and metabolic state. We employed the mouse myoblasts C2C12 differentiation model to test the regulatory effect of plasma exposure at several levels: (1) cell morphology; (2) functional activity of mitochondria; (3) expression levels of several mitochondria regulators, i.e., Atgl, Pgc1b, and miR-378a-3p. Existing databases were used to computationally predict and analyze mir-378a-3p potential targets. We show that short-term exposure to OB or OBD patients' plasma is sufficient to affect C2C12 properties. In fact, the expression of genes that regulate skeletal muscle differentiation and growth was downregulated in both OB- and OBD-treated cells, maximal mitochondrial respiration rate was downregulated in the OBD group, while in the OB group, a metabolic switch to glycolysis was detected. These alterations correlated with a decrease in ATGL and Pgc1b expression in the OB group and with an increase of miR-378a-3p levels in the OBD group.

Heparinase treatment of heparin-contaminated plasma from coronary artery bypass grafting patients enables reliable quantification of microRNAs.

BACKGROUND: microRNAs have recently been identified as powerful biomarkers of human disease. Reliable polymerase chain reaction (PCR)-based quantification of nucleic acids in clinical samples contaminated with polymerase inhibitor heparin requires deheparinization. However, the effects of deheparinization procedure on quantification of nucleic acids remain largely unknown. The aim of this study was to determine whether the deheparinization procedure completely eliminates the inhibition of amplification, while maintaining RNA integrity and technical variability of the measured microRNA levels. METHODS: Heparinized plasma from 9 patients undergoing coronary artery bypass grafting (CABG) and the heparin-free plasma from 58 rats were spiked with a synthetic RNA oligonucleotide and total RNA was extracted. The RNA solutions were then treated with heparinase I to remove contaminating heparin prior to reverse transcription. Levels of synthetic spike-in RNA oligonucleotide, as well as endogenous hsa-miR-1-3p and hsa-miR-208a-3p, were measured using quantitative reverse transcription PCR (RT-qPCR). The amplification efficiency and presence of inhibitors in individual samples were directly determined using calibration curves. RESULTS: In contrast to RNA samples from rat plasma, RNA samples derived from the CABG patient plasma contained inhibitors, which were completely eliminated by treatment with heparinase. The procedure caused a decrease in the amount of detected RNA; however, the technical variability of the measured targets did not change, allowing for the quantification of circulating endogenous hsa-miR-1-3p and hsa-miR-208a-3p in the plasma of CABG patients. CONCLUSIONS: The heparinase treatment procedure enables utilization of RT-qPCR for reliable microRNA quantification in heparinized plasma.

Brain functional connectivity network breakdown and restoration in blindness.

OBJECTIVE: To characterize brain functional connectivity in subjects with prechiasmatic visual system damage and relate functional connectivity features to extent of vision loss. METHODS: In this case-control study, resting-state, eyes-closed EEG activity was recorded in patients with partial optic nerve damage (n = 15) and uninjured controls (n = 13). We analyzed power density and functional connectivity (coherence, Granger causality), the latter as (1) between-areal coupling strength and (2) individually thresholded binary graphs. Functional connectivity was then modulated by noninvasive repetitive transorbital alternating current stimulation (rtACS; 10 days, 40 minutes daily; n = 7; sham, n = 8) to study how this would affect connectivity networks and perception. RESULTS: Patients exhibited lower spectral power (p = 0.005), decreased short- (p = 0.015) and long-range (p = 0.033) coherence, and less densely clustered coherence networks (p = 0.025) in the high-alpha frequency band (11-13 Hz). rtACS strengthened short- (p = 0.003) and long-range (p = 0.032) alpha coherence and this was correlated with improved detection abilities (r = 0.57, p = 0.035) and processing speed (r = 0.56, p = 0.049), respectively. CONCLUSION: Vision loss in the blind is caused not only by primary tissue damage but also by a breakdown of synchronization in brain networks. Because visual field improvements are associated with resynchronization of alpha band coherence, brain connectivity is a key component in partial blindness and in restoration of vision.

Noninvasive transorbital alternating current stimulation improves subjective visual functioning and vision-related quality of life in optic neuropathy.

BACKGROUND: Noninvasive repetitive transorbital alternating current stimulation (rtACS) can improve visual field size in patients with optic nerve damage, but it is not known if this is of subjective relevance. We now assessed patient reported outcomes to determine the association between visual field changes and vision-related quality of life (QoL). METHODS: Patients having visual field impairments long after optic nerve damage (mean lesion age 5.5 years) were randomly assigned to a rtACS (n = 24) or sham stimulation group (n = 18). Visual fields and patient reported outcome measures (vision-related QoL: National Eye Institute Visual Function Questionnaire, NEI-VFQ and health-related QoL: Short Form Health Survey, SF-36) were collected before and after a 10-day treatment course with daily sessions of 20 to 40 minutes. The primary outcome measure was the percent change from baseline of detection ability (DA) in defective visual field sectors as defined by computer-based high resolution perimetry (HRP). Secondary outcome parameters included further HRP parameters as well as static and kinetic perimetry results. Changes in QoL measures were correlated with changes in primary and secondary outcome measures in both groups. RESULTS: DA increase in the defective visual field was significantly larger after rtACS (41.1 ± 78.9%, M ± SD) than after sham stimulation (13.6 ± 26.3%), P < 0.05. While there was a significant increase of DA in the whole tested HRP visual field after rtACS (26.8 ± 76.7%, P < 0.05), DA in sham-stimulation patients remained largely unchanged (2.7 ± 20.2%, ns). Results of secondary outcome measures (static and kinetic perimetry) provided further evidence of rtACS efficacy. Improvements in NEI-VFQ subscale "general vision" were observed in both groups but were larger in the rtACS group (11.3 ± 13.5, Z = -3.21, P < 0.001) than in the sham group (4.2 ± 9.4, Z = -1.73, P < 0.05) with a significant difference between groups (Z = -1.71, P < 0.05). DA change and some NEI-VFQ domains were correlated (r = 0.29, P < 0.05), but no significant correlations were observed between DA and SF-36 results. CONCLUSIONS: rtACS facilitates vision restoration after unilateral, long-term optic nerve lesion as assessed both by objective DA changes and improvements in some NEI-VFQ subscales. Both were positively but low correlated, which suggests that factors other than visual field size also contribute to improved vision-related QoL.

Repetitive transorbital alternating current stimulation in optic neuropathy.

BACKGROUND: Visual field defects after optic nerve damage typically show a limited capacity for spontaneous and treatment-induced recovery. OBJECTIVE: Repetitive transorbital alternating current stimulation (rtACS) was applied to the damaged optic nerve to evaluate visual functions after stimulation. METHODS: A 27-years-old male patient suffering left optic nerve atrophy with nearly complete loss of vision 11 years after atypical traumatic damage was treated transorbitally with biphasic 10-15 pulse trains of rtACS (10-30 Hz, < 600 µA, 30-40 min daily for 10 days) which produced phosphenes. RESULTS: After rtACS treatment detection ability of super-threshold stimuli increased from 3.44% to 17.75% and mean perimetric threshold from 0 dB to 2.21 dB at final diagnostics. CONCLUSION: This improvement of vision may be due to increased neuronal synchronization, possibly involving strengthening of synaptic transmission along the central visual pathway.

Non-invasive alternating current stimulation induces recovery from stroke.

BACKGROUND: Recovery of post-stroke deficits can be achieved by modulating neuroplasticity with non-invasive brain stimulation. To evaluate potential effects of repetitive transorbital alternating current stimulation (rtACS) on stroke recovery we carried out a randomized, drug-controlled clinical trial. METHODS: Ninety-eight patients that had suffered ischemic stroke 21.4 months earlier were randomly assigned to either group D (n=30) receiving conventional drug therapy, group ACS (n=32) treated for 12 days with rtACS, or group D/ACS (n=36) receiving combined drug therapy/rtACS. Stroke severity level (SSL) was assessed by the NIH-NINDS stroke scale before and after treatment and at a 1-month follow-up to evaluate motor impairments (weakness, ataxia), sensory loss, visual field defects, and cortical deficits (aphasia, neglect). At each time point standard EEG recordings (10-20 system) were conducted. RESULTS: Before therapy SSL was moderate (9.18 ± 0.78) without significant group difference (F =0.86, p=0.43). After 12 days of treatment, SSLs of groups ACS and D/ACS significantly improved by 22.5% and 25.1% over baseline, respectively, with no such change in the control group D (+3%). SSL improvements were mainly due to recovery of motor, sensory, and speech functions. After 1-month follow-up, an additional improvement of 9.7% and 9.4% was seen for the group ACS and D/ACS which led to a total change of +32.3% and +34.7% over baseline. EEG recordings revealed greater interhemispheric synchrony between both temporal lobes which were positively correlated with clinical outcome. CONCLUSIONS: Non-invasive rtACS applied to post-stroke patients can modulate brain plasticity and induce recovery from neurological deficits long after the early post lesion recovery is over.