The electrophysiological examinations in patients with HMSN and ALS diseases showed signs of severe peripheral denervation with reinnervation, but the patient with cachexia due to malnutrition alone displayed normal EMG and ENeG findings. Methods Muscle Biopsy Biopsy specimens from the patient with cancer cachexia were obtained from the left tibialis anterior muscle

using the percutaneous conchotome method. The biopsy was dissected free of fat and connective tissues. One portion was frozen in Perifosine in vitro isopentane chilled with liquid nitrogen and stored at -160 °C for morphological analyses. Small bundles of 25-50 fibers were dissected from another biopsy specimen and membrane permeabilized Inhibitors,research,lifescience,medical (8). The muscle bundles were treated with sucrose, a cryo-protectant, for 1-2 weeks for long-term storage (9). Histopathology and electron microscopy The frozen samples were used for histopathology and sections were stained with hematoxylin Inhibitors,research,lifescience,medical and eosin, Gomori’s trichrome, and reacted for ATPases with preincubations at pH 4.3. and 10.4, NADH tetrazolium reductase, cytochrome-c-oxidase + succinate dehydrogenase and immunostained for fetal, neonatal, fast and slow myosin heavy chains (MyHCd, MyHCn, MyHCf and MyHCs; Novocastra, Newcastle-upon-Tyne, UK). The lesser diameters of type I and II fibers were measured in ATPase 4.3 stained sections using a computerized muscle biopsy analyser (Muscle

Biopsy Inhibitors,research,lifescience,medical Surveyor®; PIT Oy, Turku, Finland). For electron microscopy (EM) small pieces were routinely fixed in 3% phosphate buffered glutaraldehyde, post-osmicated, dehydrated and embedded in epon. Thin sections were double stained with uranyl acetate and lead citrate and examined Inhibitors,research,lifescience,medical in a JEOL JEM 1200 electron microscope. Single muscle fiber experimental procedure On the day of an experiment, Inhibitors,research,lifescience,medical a fiber segment length of 1 to 2 mm was left exposed to the solution between connectors leading to a force transducer (model 400A, Aurora Scientific) and a lever arm system (model 308B, Aurora Scientific) (10). The total compliance of the attachment system was carefully controlled and remained similar for all the single muscle fibers

tested (6 ± 0.4% of fiber length). The apparatus was mounted on the stage of an inverted microscope (model IX70; Olympus). While the fiber segments were in relaxing solution, sarcomere length was set to 2.75-2.85 μm by adjusting the overall segment length only (8). The sarcomere length was controlled during the experiments using a high-speed video analysis system (model 901A HVSL, Aurora Scientific). The fiber segment width, depth and length between the connectors were measured (8). Fiber cross-sectional area (CSA) was calculated from the diameter and depth, assuming an elliptical circumference, and was corrected for the 20% swelling that is known to occur during skinning (10). The maximum force normalized to fiber cross-sectional area (CSA) was measured in each muscle fiber segment (8).