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. 2022 Jul 5;12(13):e4461.
doi: 10.21769/BioProtoc.4461.

Experimental Models for Cold Exposure of Muscle in vitro and in vivo

Tiril Schjølberg  1 , Lucia Asoawe  1 , Solveig Krapf  2 , Arild C Rustan  2 , Hege G Thoresen  2   3 , Fred Haugen  1
Affiliations

Experimental Models for Cold Exposure of Muscle in vitro and in vivo

Tiril Schjølberg et al. Bio Protoc. .

Abstract

Work in cold environments may have a significant impact on occupational health. In these and similar situations, cold exposure localized to the extremities may reduce the temperature of underlying tissues. To investigate the molecular effects of cold exposure in muscle, and since adequate methods were missing, we established two experimental cold exposure models: 1) In vitroexposure to cold (18°C) or control temperature (37°C) of cultured human skeletal muscle cells (myotubes); and 2) unilateral cold exposure of hind limb skeletal muscle in anesthetized rats (intramuscular temperature 18°C), with contralateral control (37°C). This methodology enables studies of muscle responses to local cold exposures at the level of gene expression, but also other molecular outcomes. Graphical abstract.

Keywords: Cold exposure; In vitro; Myotubes; Protein expression; Skeletal muscle; mRNA.

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Conflict of interest statement

Competing interestsThe authors have no financial and non-financial competing interests.

Figures

Figure 1.
Figure 1.. Setup of in vitroexposure of myotubes in CO 2incubator with parallel exposure of cells to either warm or cold temperature.
Thermal block (highlighted with dashed lines, either blue indicating cold exposure or red indicating control conditions of 37°C) temperatures are controlled via connected water circulators (Control = red tubing; cold = blue tubing). Culturing conditions (the actual exposure temperature) are monitored with a thermocouple probe and PicoLog software. Styrofoam insulation is not shown but should be mounted around both tubing and thermos blocks. Figure created in BioRender (biorender.com).
Figure 2.
Figure 2.. Thermo block.
A) Back and front sides of the aluminum thermo block (light blue), strung with serpentine copper tubes (brown) and with grooves milled into it (dark blue), allowing a tight contact to form between the thermo block and the culture plate wells. B) The copper tubing and its dimensions (mm) in the thermo block.
Figure 3.
Figure 3.. Setup of in vivocold exposure.
Isoflurane anesthetized (mask) rat placed in supine position on a heating pad (red), with left hind limb fixed with surgical tape to a cooling plate (blue); the right hind limb serves as a contralateral control. Rectal probe and vaporizer are not shown. Figure created in BioRender (biorender.com).
Figure 4.
Figure 4.. Temperature in thermo block and culture wells after 30 min at six different water circulator temperatures.
Mean + SEM values are shown for 18 culture wells in three 6-well plates (N = 18).
Figure 5.
Figure 5.. Linear relation (R 2= 0.99) between culture well and water circulator temperature.
Mean + SEM values are shown for 18 culture wells in three 6-well plates (N = 18).
None
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