The overall goal of this experiment with a broad span of methods is to examine muscle aerobic capacity, muscle protein expression, glucose tolerance, strength, and power in elderly people performing short term progressive resistance training. The methods used in this study, they are not unique but by combining them with a broad spectrum of methods and applying them to studies of strength training in elderly, we were able to show very important reasons not only concerning improvements in strength, which was expected, but also in muscle aerobic capacity, in insulin sensitivity as well as other aspects in muscle tissue. The value of the methods we have used is great regarding therapy with strength training for elderly people who are physically inactive.
And the methods we use are, for example, following strength, power, glucose tolerance, and protein expressions. These methods, you can use them into rehabilitation, strength training for old people, among the elderlies. But in addition to this, you can even use them in other kind of context such as sports physiology.
Generally individuals new to these methods will struggle from the multi-step process in examining protein expression in the muscle. Also for the elderly subjects will struggle from obtaining real maximum value for strength and power. These methods are developed in order to follow the changes in the muscle with power, energy, concentration, in science concentration, and other factors of importance with strength training.
Begin by asking the subject to sit on the bench of an isokinetic dynamometer. Fix the subject's trunk with straps over the shoulders and hips. And securely strap the subject's shank to the dynamometer shaft with two straps, one below the knee and one above the ankle.
Then align the knee joint axis with the rotational center of the dynamometer shaft. Set the range of motion between 90 degrees and 15 degrees. After the subject is secured, ask them to perform a total of eight trials alternating between four maximal voluntary eccentric extensions and four maximal voluntary concentric knee extensions with the right leg at a constant angular velocity of 30 degrees per second.
Specifically for the eccentric task, ask the subject to resist the dynamometer shaft with maximal effort through the whole movement from the 15 degree to 90 degree knee angle. While in the concentric task, ask the subject to press the lower leg in the dynamometer shaft in a knee extension as hard as possible throughout the whole motion range. Next, allow a four-minute rest after the dynamic recordings.
Then, assess the static maximal voluntary contraction torque four times at a 65-degree knee angle by asking the subject to kick as fast and as hard as they can against the dynamometer shaft which should be fixed at 65 degrees. In the software program for muscle strength analysis register the highest peak torque in the eccentric and concentric recordings respectively for each subject and the highest strength value among the four static trials. Following the eccentric, concentric, and static measurements, record the highest value obtained from each task for each subject.
Then, move the cursor to the 7.5 newton metre value on the Y scale to obtain the position for zero milliseconds in order to measure the rate of forced development during zero to 30 milliseconds and zero to 200 milliseconds in the highest value found among the static trials. Finally, for the pretest assessment, set the cursor on the 30 millisecond value and write down the value showing the raise in newton meters at 30 milliseconds. To take a muscle biopsy on test day two, start by injecting one to two milliliters of local anesthesia subcutaneously and into the fascia.
After a couple of minutes, make and incision with a small scalpel through the skin and fascia approximately one third of the distance from the patella to the anterior superior iliac spine. Then, take a muscle biopsy from the middle portion of the thigh muscle, vastus lateralis, using a conchotome. On test day three perform the oral glucose tolerance test or OGTT, on subjects who have fasted overnight and have not done any strenuous exercise on the testing day or the day before.
For the test, have the subject lie in a supine position. Take four milliliter blood samples via venous cannula in the antecubital vein 15 minutes before and just prior to the intake of glucose followed by 15, 30, 60, 90, and 120 minutes after the ingestion of the glucose. Use the samples to perform standard glucose level tests.
Begin by freeze drying the muscle sample in a Lifelizer at a pressure below 10 to the minus one millibar for 12 hours. Dissect it using a needle and forceps under a light microscope so that it is free of blood and connective tissue. And store it at minus 80 degrees celsius.
Then, place a scoop of 0.5 millimeter zirconium oxide beads in each tube with the muscle. Add 80 microliters per milligram of ice cold buffer. And homogenize for two times one minute at speed step seven to eight and four degrees celsius.
Centrifuge the homogenate for 10 minutes at 10, 000 times G.Transfer the remaining supernatant to new tubes and discard the pellet containing the structural proteins. Spectrophotometrically determine the protein concentration in the supernatant with a commercially available kit using a plate reader at 660 nanometers. Then dilute the samples with 2x Laemmli sample buffer and homogenizing buffer to a final protein concentration of 1.5 micrograms per microliter.
Heat them to 95 degrees celsius for five minutes to denature the proteins. Store the diluted samples at minus 20 degrees celsius prior to analysis. For native polyacrylamide gel electrophoresis, of PAGE, load 30 micrograms of protein from each sample into 18 well precast gradient cells.
And perform electrophoresis at 300 volts for 30 minutes on ice. Next, equilibrate the gel and transfer buffer for 30 minutes at four degrees celsius. Transfer proteins to polyvinylidene fluoride membranes with 0.2 micron pore sizes at a constant current of 300 milliamps for three hours at four degrees celsius.
To confirm equal loading and transfer stain the membranes with a total protein stain. The membrane is separated for each target protein. Then, block the membrane for one hour at room temperature in Tris buffered saline containing 5%nonfat milk.
Add primary antibodies diluted in TBS containing 2.5%nonfat milk and supplemented with 0.1%Tween-20. And then incubate the membranes overnight. Following primary antibody incubation wash the membranes with TBSTM, and incubate with secondary antibodies conjugated with horseradish peroxidase for one hour at room temperature.
Wash again with TBSTM followed by four additional five-minute washes with TBS. Finally, apply six to 12 mL of chemiluminescent substrate to the membrane for five minutes. Place the membrane between two transparent plastic sheets.
Then in front of a CCD camera blocking external light take serial exposures using a chemiluminescent camera filter. Compared to the CON group, the RET group showed improvement in static, eccentric, and concentric strength with eight to 12%Further, the dynamometer measurements also showed for the rate of forced development an increase of 52%at the initial zero to 30 milliseconds in the RET group. From the muscle biopsy samples, histochemistry indicated that the amount of fiber type IIa increased, and there was a trend to a decrease in 2x for the RET group from pre to post.
More from the muscle biopsy samples, protein content related to the signaling of muscle protein synthesis showed a rise of 69%for both Akt and mTOR in the RET group. Analyses also proved among mitochondrial proteins an increase of about 30%for OXPHOS Complex II and citrate synthase and of 90%for complex IV only in the RET group. Further, the RET group's results from the glucose tolerance test showed improved blood glucose, both in blood values after two hours and for the area under the curve.
The measurements done in this study were done on three different separate days for each subject and with one day's rest in between. That means the testing could be done in one week altogether for one subject. In addition to that, we have the muscle analysis, and Western Blot analysis, which takes another two days.
While attempting, in these kind of procedures or projects, it's important to remember that you should have a day of rest between each test day. And for the subjects, of course, it's very important to know that they shouldn't do any kind of rigorous exercise the same day and the day before the test day, or each test day. By using the methods we have in the project one could also measure the maximum strength in the abdominal, in the back, in the arm muscles and see how maximal strength may improve, as well, in those groups of muscles by short term resistance training.
Don't forget that when working with elderly people in strength training progress one should maintain or monitor their health status and also progressively increase the chain load in a manner that they will tolerate and also to keep their wellbeing.
