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Lionikas1 project protocol

Physiological characteristics and responses to endurance training in 6 inbred strains of mice   (2012)

Kilikevicius A, Lionikas A
With: Venckunas T, Zelniene R, Carroll AM, Lionikaite S, Ratkevicius A




Lionikas1 Protocol

Project protocol - Contents

Workflow and sampling

Step
Procedure
Equipment
Data collected
1
 Mice divided using split-litter design into control and endurance exercise groups
-
-
2 Body weight measured starting 1 wk before commencement of experiment; then weighed at 1-wk intervals until the end of testing scales body weight
3
 Swim training sessions conducted 5 days a week for 5 wks
Swimming arena
-
4 Swimming endurance testing Swimming arena -
5 Mice enthanized, tissues harvested and assessed Dissection kit, scales, cryotome, microscope, spectrophotometer
Weight of ventricles and hindlimb muscles; length of femur; muscle histology; mitochondrial enzyme activity

Equipment and supplies

  • Scales (Philips HR 2385, Vienna AUSTRIA)
  • Swimming arena (see details below)
  • Electronic balance (KERN ABS 80-4, Balingen GERMANY)
  • Dissection kit
  • Caliper (Limit electronic sliding, Alingsas SWEDEN)
  • Cryotome (Leica CM1850UV, Leica Microsystems Nussloch GmbH, Nussloch GERMANY)
  • Microscope with digital camera
  • ImageJ software (NIH v 1.43, Bethesda MD)
  • Spectrophotometer

Reagents and solutions

  • Isopentane pre-chilled with liquid nitrogen
  • Myosin ATPase stain
  • Lysis buffer [50 mM Tris-HCl, 100 mM KHPO4, 2 mM EDTA, 0.2% (wt/vol) bovine serum albumin; pH 7.0]
  • Bradford protein assay reagent
  • Coenzyme A-5,5'-Dithiobis(2-nitrobenzoic acid) [DTNB]
  • Citrate synthase standard (porcine; C3260-200UN, Sigma-Aldrich, Gillingham Dorset UK)
  • Citrate synthase reaction reagent [100 mM triethanolamine-HCl, 100 µM DTNB, Triton-S (0.25% vol/vol), 0.5 mM oxaloacetate, 0.31 mM acetyl CoA; pH 8.0
  • β-hydroxyl-coenzyme (CoA) dehydrogenase reaction reagent [100 mM tetrasodiumpyrophosphate, 0.23 mM NADH, 0.24 mM acetoacetyl CoA; pH 7.3

Procedure: Test groups and body weight monitoring

    1. Mice are divided using a split-litter design into control and endurance exercise groups (groups are balanced based on date of birth and body weight).
    2. Control mice are treated in the same way as the exercise group except for swimming training (control mice are subjected to water 2 min per session to mimic stress associated with water immersion).
    3. Mice are weighed to the closest gram starting 1 wk prior to training.
    4. Mice are then monitored at 1-wk intervals throughout the study (body weights recorded).

Procedure: Swimming endurance training and testing

    1. The swimming arena consists of 25 bowls (15 x 14 x 25 cm) filled with ~21 cm water (35 +/- 1°C), placed in a 100 x 100 cm water tank, wherein the water level is adjusted to about half the height of the plastic bowls. Water temperature is monitored and maintained during each session.
    2. Mice swim once a day, 5 days a week for 5 wks. (To minimize floating, coats are soaked with liquid soap prior to swimming).
    3. Duration of training sessions is progressively increased (for mice in the endurance exercise group only) from 15 to 150 min using a protocol described by Evangelista et al., 2003. Initially, the duration of swimming is lengthened by a 15-min increment every training session until 90 min is reached, and then by a 10-min increment until 150 min is reached.
    4. Peak duration is maintained until the end of training. (Mice exhibiting signs of exhaustion during the training session are removed from the water and allowed to rest for 3 min in their home cage before resuming the session. If this happens a second time, a 4-min rest is allowed. The session is discontinued if the signs of exhaustion are noted for a third time.)
    5. To measure endurance capacity and the effects of endurance training, half of the exercise group and half of the control group are randomly assigned to perform swimming until exhaustion.
    6. Exhaustion is declared when a mouse changes its typical swimming pattern into an intensive rotation, exhibits rising and vigorous kicking movements with forelimbs, or the inability to maintain its head above the surface of the water for >7 s.

Procedure: Heart and femur sampling

    1. Mice are enthanized by cervical dislocation.
    2. Cardiac ventricles are excised immediately, cleaned of excess blood, and weighed to 0.1 mg accuracy on an electronic balance.
    3. The following hindlimb (left) muscles are dissected and weighed similarly: tibialis anterior, extensor digitorum longus, gastrocnemius, soleus, and quadriceps femoris.
    4. Skeletal muscles are then snap frozen in isopentane and stored at -80°C for histology and mitochondrial enzyme activity (below).
    5. Adherent tissue is removed from the femur, and the length measured with an accuracy of 0.1 mm with a caliper.

Procedure: Skeletal muscle histology and image analysis

    1. Samples are sorted within-strain by soleus weight and 11-12 samples closest to the mean are selected for histological analyses. (Soleus muscle is chosen because the number of fibers can be accurately assessed at the mid portion of the muscle.)
    2. Transverse sections (10 µm) are cut with a cryotome at -20°C.
    3. Sections are subjected to myosin ATPase staining following acid-preincubation.
    4. Cross-sectional images are taken at x5 and x20 objective for fiber count (total fiber count, percent of type I fibers, intermediately stained fiber count) and cross-sectional area (measured in 25 type I and type IIa fibers of each sample).
    5. Muscle fiber traits are analyzed using ImageJ software.

Procedure: Mitochondrial enzyme activity

    1. Gastrocnemius samples are placed in 10 volumes of ice cold lysis buffer and homogenized.
    2. Homogenates are shaken for 60 min and centrifuged at 13K g for 10 min.
    3. Supernatants are taken and protein concentration is measured using the Bradford assay.
    4. Spectrophotometric measurements are carried out at room temperature as follows:
    5. Citrate synthase: Reaction reagent (1 mL) includes 10 µL of muscle homogenate; porcine citrate synthase is used as a standard for assay calibration; molar extinction coefficient is 13,600 L/mol/Cm DTNB at 412 nm.
    6. β-hydroxyl-coenzyme (CoA) dehydrogenase: Reaction reagent (1 mL) includes 20 µL of muscle homogenate; molar extinction coefficient is 63,000 L/mol/Cm for nicotinamide-adenine dinucleotide (NADH) at 340 nm.

Data collected by investigator

  • body weight
  • duration of endurance swimming
  • femur length
  • ventricle (heart) weight
  • skeletal muscle morphology
  • mitochondrial enzyme activity


References

    Evangelista FS, Brum PC, Krieger JE. Duration-controlled swimming exercise training induces cardiac hypertrophy in mice. Braz J Med Biol Res. 2003 Dec;36(12):1751-9. Epub 2003 Nov 17. PubMed 14666261