Femke Mathot

Chapter 7 114 0.02ms at an intensity level of 2.7mA was applied. Maximal amplitude measurements were obtained bilaterally. 33, 34 Isometric Tetanic Force (ITF) - The ITF was measured bilaterally per the protocol of Shin and colleagues. 35 The peroneal nerve and tibial muscle were exposed and the hind limb was secured to a testing platform with K-wires through the femur and ankle. The tibial tendon was secured to a clamp in anatomical position and attached to a force transducer (MDB-50; Transducer Techniques, Temecula, CA, USA) whose signals were processed using LabView (National instruments, Austin, Texas). A miniature electrode (Harvard Apparatus, Holliston, MA, USA), stimulated by a bipolar stimulator (Medtronic, Minneapolis, MN, USA) was clamped around the peroneal nerve branch of the sciatic nerve. The muscle tension and the stimulator frequency were optimized after which the maximal ITF was obtained. The tibial muscle was kept moist with warm saline. Wet tibial muscle mass - Rats were euthanized with an overdose of pentobarbital (Fatal Plus, 390 mg/mL, Vortech, Dearborn, MI, USA) intraperitoneally. Tibial muscles were harvested bilaterally and wet muscle mass was determined after removing the tendon. Histology - A three millimeter segment of both peroneal nerves of all rats were collected and placed into Trumps solution. Specimens were processed with 0.1M Phosphate Buffer, 1% Osmium tetroxide in buffer, graded series of alcohol and acetone. The samples were infiltrated in a 50%, 75% and finally 100% epoxy resin and polymerized at 65°C for 12-18 hours. Samples were cut in sections at 0.6 microns, placed on slides and stained on a warming plate with Toluidine blue for 2-2.5 minutes. The total tissue cable area (nerve area), axon area, axon count and myelin area were obtained using a Nikon Eclipse 50i microscope and Image Pro Plus Software. The N-ratio was calculated by dividing the myelinated fiber area (axon area and myelin area) by the tissue cable area. 36 Immunofluorescence - Both sciatic nerves of five randomly selected rats per group were dissected and fixed in 10% formalin for 48 hours. Nerves samples were vertically embedded in paraffin and sections from the exact middle were stained for Schwann cell marker S100 and protein gene product 9.5 (PGP9.5), a pan neuronal marker. Immunohistochemical staining was performed at the Pathology Research Core (Mayo Clinic, Rochester, MN, USA) using the Leica Bond RX stainer (Leica, Buffalo Grove, IL, USA). The S100 (rabbit polyclonal; Dako, Agilent Technologies Inc., Carpinteria, CA, USA) and PGP9.5 primary antibody (rabbit polyclonal; Dako, Agilent Technologies Inc.) were diluted to 1:5000 in Background Reducing Diluent (Dako, Agilent Technologies Inc.) and incubated for 60 minutes with the samples, prior to staining with the appropriate secondary antibody (Alexa Goat-Anti-Rabbit 488, 1:300, for S100 and Alexa Goat-Anti-Rabbit 568, 1:200 for PGP9.5) and counterstained with Hoechst 33342 (all ThermoFisher Scientific, MA, USA). Images of the stained slides were obtained with a fluorescence laser confocal microscope (Zeiss LSM 780, Carl Zeiss Surgical GmbH, Oberkochen, Germany). The mean fluorescent density of both stains was measured using ImageJ software.