Passive mechanical features of single fibers from human muscle biopsies – effects of storage

<p>Abstract</p> <p>Background</p> <p>The purpose of this study was to investigate the effect of storage of human muscle biopsies on passive mechanical properties.</p> <p>Methods</p> <p>Stress-strain analysis accompanied by laser diffraction assisted sarcomere length measurement was performed on single muscle fibres from fresh samples and compared with single fibres from stored samples (-20°C, 4 weeks) with the same origin as the corresponding fresh sample. Basic morphological analysis, including cross sectional area (CSA) measurement, fibre diameter measurement, fibre occupancy calculation and overall morphology evaluation was done.</p> <p>Results</p> <p>Statistical analysis of tangent values in stress-strain curves, corresponding to the elastic modulus of single muscle fibres, did not differ when comparing fresh and stored samples from the same type of muscle. Regardless of the preparation procedure, no significant differences were found, neither in fibre diameter nor the relation between muscle fibres and extra-cellular matrix measured under light microscopy.</p> <p>Conclusion</p> <p>We conclude that muscle fibre structure and mechanics are relatively insensitive to the storage procedures used and that the different preparations are interchangeable without affecting passive mechanical properties. This provides a mobility of the method when harvesting muscle biopsies away from the laboratory.</p

Reactive Oxygen Species Modulate the Barrier Function of the Human Glomerular Endothelial Glycocalyx

Reactive oxygen species (ROS) play a key role in the pathogenesis of proteinuria in glomerular diseases like diabetic nephropathy. Glomerular endothelial cell (GEnC) glycocalyx covers the luminal aspect of the glomerular capillary wall and makes an important contribution to the glomerular barrier. ROS are known to depolymerise glycosaminoglycan (GAG) chains of proteoglycans, which are crucial for the barrier function of GEnC glycocalyx. The aim of this study is to investigate the direct effects of ROS on the structure and function of GEnC glycocalyx using conditionally immortalised human GEnC. ROS were generated by exogenous hydrogen peroxide. Biosynthesis and cleavage of GAG chains was analyzed by radiolabelling (S35 and 3H-glucosamine). GAG chains were quantified on GEnC surface and in the cell supernatant using liquid chromatography and immunofluorescence techniques. Barrier properties were estimated by measuring trans-endothelial passage of albumin. ROS caused a significant loss of WGA lectin and heparan sulphate staining from the surface of GEnC. This lead to an increase in trans-endothelial albumin passage. The latter could be inhibited by catalase and superoxide dismutase. The effect of ROS on GEnC was not mediated via the GAG biosynthetic pathway. Quantification of radiolabelled GAG fractions in the supernatant confirmed that ROS directly caused shedding of HS GAG. This finding is clinically relevant and suggests a mechanism by which ROS may cause proteinuria in clinical conditions associated with high oxidative stress. © 2013 Singh et al.published_or_final_versio

Test-retest reliability of knee kinesthesia in healthy adults

<p>Abstract</p> <p>Background</p> <p>Sensory information from mechanoreceptors in the skin, muscles, tendons, and joint structures plays an important role in joint stability. A joint injury can lead to disruption of the sensory system, which can be measured by proprioceptive acuity. When evaluating proprioception, assessment tools need to be reliable. The aim of this study was to assess the test-retest reliability of a device designed to measure knee proprioception.</p> <p>Methods</p> <p>Twenty-four uninjured individuals (14 women and 10 men) were examined with regard to test-retest reliability of knee kinesthesia, measured by the threshold to detection of passive motion (TDPM). Measurements were performed towards extension and flexion from the two starting positions, 20 degrees and 40 degrees knee joint flexion, giving four variables. The mean difference between test and retest together with the 95% confidence interval (test 2 minus test 1), the intraclass correlation coefficient (ICC_2,1), and Bland and Altman graphs with limits of agreement, were used as statistical methods for assessing test-retest reliability.</p> <p>Results</p> <p>The intraclass correlation coefficients ranged from 0.59 to 0.70 in all variables except one. No difference was found between test and retest in three of the four TDPM variables. TDPM would need to decrease between 10% and 38%, and increase between 17% and 24% in groups of uninjured subjects to be 95% confident of detecting a real change. The limits of agreement were rather wide in all variables. The variables associated with the 20-degree starting position tended to have higher intraclass correlation coefficients and narrower limits of agreement than those associated with 40 degrees.</p> <p>Conclusion</p> <p>Three TDPM variables were considered reliable for observing change in groups of subjects without pathology. However, the limits of agreement revealed that small changes in an individual's performance cannot be detected. The higher intraclass correlation coefficients and the narrower limits of agreement in the variables associated with the starting position of 20 degrees knee joint flexion, indicate that these variables are more reliable than those associated with 40 degrees. We, therefore, recommend that the TDPM be measured with a 20-degree starting position.</p

On The Rate and Extent of Drug Delivery to the Brain

To define and differentiate relevant aspects of blood–brain barrier transport and distribution in order to aid research methodology in brain drug delivery. Pharmacokinetic parameters relative to the rate and extent of brain drug delivery are described and illustrated with relevant data, with special emphasis on the unbound, pharmacologically active drug molecule. Drug delivery to the brain can be comprehensively described using three parameters: Kp,uu (concentration ratio of unbound drug in brain to blood), CLin (permeability clearance into the brain), and Vu,brain (intra-brain distribution). The permeability of the blood–brain barrier is less relevant to drug action within the CNS than the extent of drug delivery, as most drugs are administered on a continuous (repeated) basis. Kp,uu can differ between CNS-active drugs by a factor of up to 150-fold. This range is much smaller than that for log BB ratios (Kp), which can differ by up to at least 2,000-fold, or for BBB permeabilities, which span an even larger range (up to at least 20,000-fold difference). Methods that measure the three parameters Kp,uu, CLin, and Vu,brain can give clinically valuable estimates of brain drug delivery in early drug discovery programmes

High glucose causes dysfunction of the human glomerular endothelial glycocalyx

The endothelial glycocalyx is a gel-like layer which covers the luminal side of blood vessels. The glomerular endothelial cell (GEnC) glycocalyx is composed of proteoglycan core proteins, glycosaminoglycan (GAG) chains, and sialoglycoproteins and has been shown to contribute to the selective sieving action of the glomerular capillary wall. Damage to the systemic endothelial glycocalyx has recently been associated with the onset of albuminuria in diabetics. In this study, we analyze the effects of high glucose on the biochemical structure of the GEnC glycocalyx and quantify functional changes in its protein-restrictive action. We used conditionally immortalized human GEnC. Proteoglycans were analyzed by Western blotting and indirect immunofluorescence. Biosynthesis of GAG was analyzed by radiolabeling and quantified by anion exchange chromatography. FITC-albumin was used to analyze macromolecular passage across GEnC monolayers using an established in vitro model. We observed a marked reduction in the biosynthesis of GAG by the GEnC under high-glucose conditions. Further analysis confirmed specific reduction in heparan sulfate GAG. Expression of proteoglycan core proteins remained unchanged. There was also a significant increase in the passage of albumin across GEnC monolayers under high-glucose conditions without affecting interendothelial junctions. These results reproduce changes in GEnC barrier properties caused by enzymatic removal of heparan sulfate from the GEnC glycocalyx. They provide direct evidence of high glucose-induced alterations in the GEnC glycocalyx and demonstrate changes to its function as a protein-restrictive layer, thus implicating glycocalyx damage in the pathogenesis of proteinuria in diabetes. Copyright © 2011 the American Physiological Society.link_to_subscribed_fulltex