3.2.1 Dissection procedures
Skin was removed from the lower limb and under a dissecting microscope (Leica, ZOOM 2000TM Z45V, China) the proximal tendon of the EDL was located by clearing away the surrounding tissues. Fine tipped forceps were then inserted under the proximal EDL tendon and size 2.0 surgical silk (LA-55G Ethicon, Johnson & Johnson, NSW, Australia) was fed through and then tied securely around the tendon to form a “stopper knot”. Size 4.0 surgical silk was then tied to the tendon, distal to the stopper knot, which then formed the ties that would be used to secure the muscle to the contractile setup. The same isolation and tying technique was then performed for the distal EDL insertion. The distal tendon of the EDL was then cut and the muscle was carefully dissected away from the other tissues to the point of the proximal tendon insertion.
Once fully dissected out the muscle was quickly placed into a horizontal custom-built plexiglass muscle bath containing Krebs Henseleit Ringer solution [NaCl 118 mM; KCL 4.75 mM; Na2HPO4 1 mM; MgSO47H2O 1.18 mM; NaHCO3 24.8 mM; CaCl2 2.5 mM and
D-Glucose 11.0 mM, pH 7.4]. This HCO3- based buffer solution was aerated with
carbogen (95% O2 and 5% CO2) (BOC gases, Melbourne, Australia). The temperature of
the muscle bath was maintained (see individual chapters for specific temperatures) by circulating heated water internally through the plexiglass bath and ensuring constant laboratory temperature, both of which were monitored throughout the experimental protocol. The muscle was then left in the bath to equilibrate while contralateral EDL was dissected out and placed in another muscle bath containing Krebs ringer solution bubbled with carbogen. This muscle was maintained in the bath for the same duration as the contracted. The exposed surgery sites on the mouse were wrapped in saline-soaked gauze, to prevent drying out of the tissues. The level of anaesthesia was monitored periodically while the contractile protocol was being conducted on the EDL, with additional doses of Nembutal being administered as required.
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Once the EDL contractile protocol was completed the muscle was removed from the contractile setup, blotted on filter paper (Whatman No. 1, Maidstone, UK) and the silk and excess tendon removed. The muscle was then weighed and snap frozen in liquid nitrogen. The Krebs ringer solution in the bath was drained, the bath was cleaned and fresh solution was added. The SOL muscle was then dissected and tied, using the same technique as was described previously for the EDL. The contralateral SOL was then removed and placed a muscle bath as previously described for EDL, while a sample of blood was taken and additional muscles removed. The Plantaris (PLANT), TA and GAST muscles were all removed and snap frozen before the chest cavity was opened, and a small section of diaphragm was cut close to the rib cage to allow access to the heart. The heart was then pierced with a 22 gauge catheter (Terumo, NSW, Australia) and resulting blood flow was collected (Chapter 6), or cavity blood was collected once the heart was removed (Chapter 5), which was then centrifuged at 12, 000 RPM for 2 minutes. Plasma was carefully removed, placed in a cryule and snap frozen in liquid nitrogen. The diaphragm and heart were then dissected out, blotted on filter paper to remove any excess fluid or blood, and snap frozen. The contractile protocol was then performed on the SOL, with the contracted and contralateral rested muscle being snap frozen at the conclusion of the contractile protocol. All muscle and plasma samples were then stored (-80°C) until further analysis.
3.2.2 Stimulation protocols
The general procedure for the contractile protocol performed in Chapter 4 and 6 was similar, as was the procedure for the different muscles (EDL and SOL). Thus, a description of common procedures is presented within this chapter. Chapters 4 and 6 specifically detail the stimulation frequencies, fatigue and recovery times that were utilised for individual studies.
Once the muscle had equilibrated in the bath, the proximal end of the muscle was tied to a micromanipulator, while the distal end was attached directly to an isometric force transducer (Research Grade 60-2999, Harvard Apparatus, South Natich, MA). This arrangement ensured that the direction of force mimicked that of the muscles natural direction of force development in vivo. Within the bath, the muscle was flanked by field- stimulating platinum-plate electrodes attached to a stimulator (Grass S1 stimulator,
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Quincy, MA), coupled to an amplifier (CE-1000, Crown Instruments, Elkhart, IN, USA) to ensure supramaximal stimulation. Stimulation of the muscle by the electrodes results in deflection of the force transducer (which was previously calibrated with a calibration weight of known mass), and a measurable electrical signal that is proportional to the force produced. These electrical signals were converted to a digital signal by Powerlab 4510 (ADI Instruments, Castle Hill, NSW, Australia) running Chart, Version 5.02 for Windows.
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Once secured, the optimal length (Lo) of the muscle was determined by eliciting twitch
contractions and adjusting the muscle length with the micromanipulator, until the maximum twitch force (Pt) was obtained. The muscle length was then measured and
later used to determine the optimal fibre length (Lf) with the previously established Lf to
Lo ratio for mouse of 0.44 for EDL and 0.71 for SOL (Brookes and Faulkner, 1988). A
single tetanic stimulation at 100 Hz for EDL and 80 Hz for SOL was then performed, to ensure that the knots anchoring the muscle to the contractile setup were secure. If a slip occurred at this point, or at any time throughout the experiment, the muscle length was again adjusted until optimal length was re-established. Following a three minute recovery period, the force frequency relationship was established by stimulating the muscle at frequencies ranging from 10-160 Hz, with 3 minutes rest between stimulations. The Po was determined from the greatest force produced during the force
frequency stimulations. The force produced at each stimulation frequency was then expressed as a percentage relative to the Po obtained. The sPo, which accounts for
differences in muscle CSA and length was also determined. This was done by first calculating CSA with the equation below;
CSA = Muscle Mass (mg) Fibre length x density
The fibre length was determined using the previously established Lf to Lo ratios for EDL
and SOL, and density is equal to 1.06 g/cm3 (Close, 1972).
Peak isometric twitch force was then measured by eliciting three twitch contractions (0.2ms) and calculating the mean value for the factors below;
1. Pt
2. TTP 3. ½ RT
These measures were performed as they can be used as a crude indicator of the rate of calcium release (TTP) and re-uptake (½ RT) into the sarcoplasmic reticulum. Finally, muscles were subjected to a fatigue protocol that consisted of a continuous high frequency, short duration stimulation (see Chapter 5) or intermittent stimulation at more
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physiological frequencies with recovery measures (see Chapter 6). On completion of contractile testing, muscles were quickly removed from the muscle bath, blotted on filter paper, and the tendons and surgical silk removed. The muscle was then weighed, snap frozen and stored at -80°C until further analysis.
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