689356
Rattus norvegicus
Protocols: The experimental rats were anaesthetized, mechanically ventilated and treated with α-cobratoxin for durations varying from 6 h to 14 days. Briefly, all surgery was performed under sterile techniques. 1) Precordial silver wire electrocardiogram (ECG) electrodes were implanted subcutaneously. 2) An aortic catheter (28-gauge Teflon) was inserted via the left carotid artery to record arterial blood pressure. 3) A 0.9 mm Renathane catheter was threaded into the left jugular vein to record venous blood pressure and administers parental solutions. 4) Three subcutaneous electroencephalogram needles were placed into the skull above the right and left temporal lobes and a third reference electrode was placed in the neck region. 5) Temperature was measured by a vaginal thermistor and servo regulated at 37 °C. 6) A silicon cannula was inserted in the urethra to continuously record urine output. Animals were maintained in protein and ﬂuid balance with an intra-arterial solution (0.6 ml/h) consisting of 50 ml H2O, 50 ml 0.5 N lactated Ringer solution, 1.25 g sodium oxacillin, 2.8 mg α-cobratoxin, 0.3 mg vitamin K (Synkavite) and 20 meq K+ (as KCl), as well as with an intra-venous solution (0.6 ml/h) consisting of 50 ml H2O, 50 ml 0.5 N lactated Ringer solution, 20% glucose (Baxter, Deerﬁeld, IL, USA) and 1.25 g sodium oxacillin. The sham operated controls rat underwent the same intervention as the experimental group animals but were not pharmacological paralyzed with α-cobratoxin. During surgery or at any possible irritating manipulation, the anesthetic isoflorane level was >1.5%. Isoflorane was delivered into the inspiratory gas stream by a precision mass-flow controller. After the initial surgery, isoflorane was gradually lowered over 1-2 days and maintained at < 0.5% during the remaining experimental period. Rats were ventilated through a per os coaxial tracheal cannula at 72 breaths/minute with an inspiratory and expiratory ratio of 1:2 and a minute volume of 180-200 ml. Neuromuscular blockade (NMB) was induced on the first day and maintained by continuous infusion. Mechanical ventilation was started immediately after the NMB induction. The left leg of the animal was activated for 6 hours at the shortest duration and 12 hours per day at duration 12 hours and longer throughout the experiment, using a mechanical lever arm that produces a continuous passive maximal ankle joint flexion-extension at a speed of 13.3 cycles per minute. Total RNA was isolated from the proximal part of gastrocnemius muscle using spin column purification RNAeasy (QIAGEN, Hilden, Germany). 300 ng of total RNA were extracted from the muscle samples. The proximal part was reversed transcribed to cDNA using Qscript cDNA super mix (Quanta Biosciences, Gaithersburg, Maryland, USA) and was diluted into a final volume of 100 µL. Primers were designed using the software Primer 3 (http://biotools.umassmed.edu/bioapps/primer3_www.cgi) and ordered from Sigma-Aldrich (Sigma-Aldrich, St Louis, Missouri, USA). cDNA was amplified in triplicate using MyiQ™single color real time PCR detection system (Bio-Rad Laboratories, Inc., Hercules, California, USA). Sequencing libraries were constructed using the ScriptSeq mRNA-Seq Library Preparation Kit (Epicentre).