Chapter 5: Conclusions and Future Directions
F. Future Directions
15. Genome Analysis for Predisposition
Despite undergoing the same training protocols, some individuals develop injury while others do not. Experiments such as genome-wide association studies (GWAS) or single-nucleotide polymorphism (SNP) on patients and healthy people could be useful in identifying molecular variations among individuals that may lead to a predisposition for injury or altered responses to treatments, such as pharmacologic interventions.
16.Final Conclusions
In this chapter, we have proposed several future directions to characterize temporal muscle and tendon adaptations to exercise, define beneficial and detrimental changes, understand how acute sessions of exercise lead to chronic adaptations (or
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maladaptations), and investigate potential drug treatments for altered loading states. Results from these studies will inform development of exercise training routines that minimize and prevent injury and treatment strategies for when injury does occur.
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Appendices: Experimental Protocols
Appendix 1: Rat Treadmill Protocols
EXa: Acute Exercise on Flat Treadmill
Animals undergo 2 weeks of overuse training on a -10° treadmill, followed by a single bout of exercise
Protocol Week Day Duration Tach Reading (rpm) T1.1 1 Mon 0-10 min 37 T1.2 1 Tues 0-15 min 40 T1.3 1 Wed 0-20 min 43 T1.4 1 Thurs 0-25 min 46 T1.5 1 Fri 0-30 min 49 T2.1 2 Mon 0-30 min 51 T2.2 2 Tues 0-5 min 5-40 min Build up 54 T2.3 2 Wed 0-5 min 5-45 min Build up 57 T2.4 2 Thurs 0-10 min 10-50 min Build up 60 T2.5 2 Fri 0-10 min 10-60 min Build up 63 Exercise Single bout Mon 0-60 min 37 (flat!)
EXc: Chronic Exercise on Flat Treadmill
Animals undergo 2 weeks of training followed by their full protocol for up to 8 weeks
Protocol Week Day Duration Tach Reading (rpm) T1.1 1 Mon 0-10 min 37 T1.2 1 Tues 0-15 min 37 T1.3 1 Wed 0-20 min 37 T1.4 1 Thurs 0-25 min 37 T1.5 1 Fri 0-30 min 37 T2.1 2 Mon 0-40 min 37 T2.2 2 Tues 0-45 min 37 T2.3 2 Wed 0-50 min 37 T2.4 2 Thurs 0-55 min 37 T2.5 2 Fri 0-60 min 37
EX1.1 up to 8 weeks Mon 0-60 min 37 EX1.2 up to 8 weeks Tues 0-60 min 37 EX1.3 up to 8 weeks Wed 0-60 min 37 EX1.4 up to 8 weeks Thurs 0-60 min 37 EX1.5 up to 8 weeks Fri 0-60 min 37
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Appendix 2: Rat Syringe Training
This protocol is meant to train rats to drink from a syringe for subsequent oral
administration of drugs like ibuprofen or doxycycline. We have found that the rats love children’s berry-flavored ibuprofen, but dislike doxycycline (despite being an oil
suspension, tutti fruiti flavor) due to its bitter taste. Most likely, doxycycline animals will need to be restrained and force-fed, but the ibuprofen animals will happily drink on their own. Note that all drug animals should be syringe-trained.
Day 1: Place a wooden popsicle stick that has a strip of honey on it in each cage. Rats are free to sniff and eat the honey to avoid neophobia. Once the rats have eaten the honey from the stick, create a mixture of honey and water (much more water than honey, not a very viscous solution) and draw it up into a needleless 1ml syringe. Open the lid of the cage and allow the rats to sniff and lick the syringe. Dispense a few drops of the honey solution onto the rats’ noses and paws as they sniff the syringe. As they groom this off, they will discover that it tastes delicious. Do not expect animals to freely drink from the syringe on their first day (though some might).
Day 2: Assuming all animals cleaned their honey sticks yesterday, begin with the honey- water syringe training. Similarly, allow the animals to freely explore the syringe and squirt some fluid out as they come close so they can taste it. If the rats did not clean their honey stick yesterday, again place a popsicle stick with a strip of honey into their cage.
Day 3: Repeat honey-water syringe training.
Day 4: Last day of honey-water syringe training. By this day, nearly all animals should be comfortable drinking from the syringe.
Note: For the first 2 or so doses of ibuprofen administration, rats probably will not drink from the syringe because it is a new taste; however, since they have been syringe trained, they know how to drink from the syringe, and they just need a couple doses to realize that they enjoy the taste of the ibuprofen and they will begin drinking on their own. As already mentioned, doxycycline animals likely will need restraint. Simply hold the animal and use your thumb and index finger to grasp their jaw as you gently squirt the fluid inside their cheek. Do not try to get all 0.5 ml in their mouth at once; do about half of it at a time to allow them to swallow so they don’t spit it all out.
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Appendix 3: Rat Supraspinatus Tendon and Muscle Dissection
This protocol is adapted from the Multi-Tendon protocol, 1/20/2011.
Equipment and Supplies Needed:
#11 blades (1 per shoulder) Blade holder
Hemostats
Rat toothed pick ups
Protocol:
1) Clamp the paw with hemostats (handles facing anteriorly). Externally rotate limb by flipping hemostats over and tucking underneath rat’s abdomen.
2) Using the flat portion of the blade, make an incision along the spine of the scapula and expose shoulder and arm.
3) Starting at the cephalic vein, incise up along the posterior border of the scapular spine (down to bone) and peel away the trapezius anteriorly.
4) Laterally cut away the bursa, cut the AC joint, and peel away clavicle.
5) Grab the acromion with the pick ups, lift off of the body and cut away connective tissue, then twist pick ups to break away acromion.
6) Carefully incise along the edges of the supraspinatus while peeling the muscle away from the scapula. Make vertical “lines” with your blade tip along the scapula to fully isolate the supraspinatus muscle.
7) Follow the medial border of the scapula to divide the supraspinatus from the subscapularis muscle and expose the joint.
8) Follow the humeral head with the blade to cut the other rotator cuff tendons/biceps and free the humeral head from the glenoid.
9) Cut away excess muscle from the humeral head and expose down to the elbow joint. Dislocate the elbow joint to remove the humerus from the body.
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Appendix 4: Rat Supraspinatus Muscle and Tendon Tissue Harvest for MMP Activity
Dissection Prep (day before)
1. For all samples that will be used for protein assays/MMP activity assay:
While wearing gloves (to prevent contamination), label Eppendorf tubes (lid and side of tube) with date,rat ID number, and tissue type (see key below). Each rat should have the following 3 labeled tubes:
1 right supra tendon (R-SUP-T)
1 right supra muscle (R-SUP-M)
1 right excess supra muscle (R-SUP-EM)
Place labeled tubes in Eppendorf tube holder with lids closed.
2. Label biohazard bags (1 per animal) with animal ID number and date of sacrifice. Also label an index card with this information and a place to write the animal weight. Place index card in pocket of biohazard bags.
3. Gather all equipment and materials:
Blue “diapers”
Black pad (1 per dissector)
Specimen cup for ethanol (1 per dissector)
Specimen cup for PBS
Liquid nitrogen container
PBS squirt bottle
Gauze
Rat-toothed pick-ups (1 per dissector)
Blade holder (1 per dissector)
#11 blades (at least 2 per rat)
Curved hemostats (1 per dissector)
Bone cruncher (optional)
List of rats to be sacrificed and corresponding assays
Paper towel
Scale with bowl for animal
Labeled biohazard bags (1 per animal)
Labeled Eppendorf tubes (3 per animal)
Lab notebook
Pen and permanent marker
Dissection Prep (day of)
1. Fill cup(s) with 70% ethanol. 2. Fill cup with PBS.
3. Get liquid nitrogen.
4. Cover dissection table with blue “diapers.” 5. Line CO2 chamber with paper towel.
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Dissection
1. Sacrifice animal (1 at a time if 1 dissector or up to 1 cage with 2 animals at a time for 2 independent dissectors). Confirm death with extended duration in CO2 chamber, cessation of breath, and lack of heart beat.
2. Weigh animal. Record weight in lab notebook and appropriate index card (located in pocket of biohazard bag).
3. Working quickly, dissect the right shoulder to isolate the humerus, supraspinatus tendon, and supraspinatus muscle (Appendix 2).
Note: When not using tools, store in ethanol. Swirl in PBS before using again. 4. Rinse in PBS by swishing specimen in PBS to remove excess blood and loose
hair.
For steps 5-7, diligence and patience are key to ensure tissue is as isolated as possible! 5. Place sample on black pad (bursal side down for stability). Use curved forceps to
grasp muscle and use blade to cut a muscle chunk from the curved side, avoiding cutting the tendon. Be sure to take both deep and superficial regions of the muscle. Place in properly labeled Eppendorf tube, and immediately submerge in liquid nitrogen.
6. Once muscle sample is taken, use back of blade to scrape remaining muscle from tendon. Place this remaining muscle in properly labeled Eppendorf tube, and