The effects of cold water immersion and active recovery on inflammation and cell stress responses in human skeletal muscle after resistance exercise

Abstract

Key points: Cold water immersion and active recovery are common post-exercise recovery treatments. A key assumption about the benefits of cold water immersion is that it reduces inflammation in skeletal muscle. However, no data are available from humans to support this notion. We compared the effects of cold water immersion and active recovery on inflammatory and cellular stress responses in skeletal muscle from exercise-trained men 2, 24 and 48 h during recovery after acute resistance exercise. Exercise led to the infiltration of inflammatory cells, with increased mRNA expression of pro-inflammatory cytokines and neurotrophins, and the subcellular translocation of heat shock proteins in muscle. These responses did not differ significantly between cold water immersion and active recovery. Our results suggest that cold water immersion is no more effective than active recovery for minimizing the inflammatory and stress responses in muscle after resistance exercise.

Abstract: Cold water immersion and active recovery are common post-exercise recovery treatments. However, little is known about whether these treatments influence inflammation and cellular stress in human skeletal muscle after exercise. We compared the effects of cold water immersion versus active recovery on inflammatory cells, pro-inflammatory cytokines, neurotrophins and heat shock proteins (HSPs) in skeletal muscle after intense resistance exercise. Nine active men performed unilateral lower-body resistance exercise on separate days, at least 1 week apart. On one day, they immersed their lower body in cold water (10°C) for 10 min after exercise. On the other day, they cycled at a low intensity for 10 min after exercise. Muscle biopsies were collected from the exercised leg before, 2, 24 and 48 h after exercise in both trials. Exercise increased intramuscular neutrophil and macrophage counts, MAC1 and CD163 mRNA expression (P < 0.05). Exercise also increased IL1β, TNF, IL6, CCL2, CCL4, CXCL2, IL8 and LIF mRNA expression (P < 0.05). As evidence of hyperalgesia, the expression of NGF and GDNF mRNA increased after exercise (P < 0.05). The cytosolic protein content of αB-crystallin and HSP70 decreased after exercise (P < 0.05). This response was accompanied by increases in the cytoskeletal protein content of αB-crystallin and the percentage of type II fibres stained for αB-crystallin. Changes in inflammatory cells, cytokines, neurotrophins and HSPs did not differ significantly between the recovery treatments. These findings indicate that cold water immersion is no more effective than active recovery for reducing inflammation or cellular stress in muscle after a bout of resistance exercise.

Keywords: cryotherapy; cytokines; inflammation; macrophages; neutrophils; recovery.

Figure 1. Post‐exercise changes in CD66b⁺ neutrophil infiltration, CD68⁺ macrophage infiltration, and MAC1 and CD163 mRNA expression

Data are presented as the change in the median ± interquartile range for neutrophils and CD163 mRNA, and the geometric mean ± 95% confidence interval for macrophages and MAC1 mRNA. ACT, active recovery; CWI, cold water immersion. n = 9. ^* P < 0.05 versus pre‐exercise value.

Figure 2. Representative image of immunofluorescence staining for CD66b⁺ neutrophils

A, red laminin staining of the sarcolemma; B, blue DAPI staining of nuclei; C, green staining for CD66b; D, merged images. Arrows indicate CD66b⁺ neutrophils. Scale bar represents 50 μm. n = 9. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 3. Representative image of immunofluorescence staining for CD68⁺ macrophages

A, red laminin staining of the sarcolemma; B, blue DAPI staining of nuclei; C, green staining for CD68; D, merged images. Arrows indicate CD68⁺ macrophages. Scale bar represents 50 μm. n = 9. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 4. Post‐exercise changes in expression of IL1β, TNF, IL6 and CCL2 mRNA

Data are presented as changes in the median ± interquartile range for IL1β, IL6 and CCL2 expression, and the geometric mean ± 95% confidence interval for TNF expression. n = 9. ^* P < 0.05 versus pre‐exercise value.

Figure 5. Post‐exercise changes in expression of CCL4, CXCL2, IL8 and LIF mRNA

Data are presented as changes in the geometric mean ± 95% confidence interval. n = 9. ^* P < 0.05 versus pre‐exercise value.

Figure 6. Post‐exercise changes in expression of GDNF and NGF mRNA

Data are presented as changes in the mean ± SD for GDNF and the geometric mean ± 95% confidence interval for NGF. n = 9. ^* P < 0.05 versus pre‐exercise value.

Figure 7. Post‐exercise changes in expression of HSP70 mRNA

Data are presented as the change in the median ± interquartile range. n = 9. ^* P < 0.05 versus pre‐exercise value.

Figure 8. Representative immunoblots and post‐exercise changes in the protein content of HSP70 and αB‐crystallin in the cytosol and cytoskeletal fraction of muscle homogenates

Data are presented as the mean ± SD. n = 9. ^* P < 0.05 versus pre‐exercise value. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 9. Intramuscular localisation of αB‐crystallin

Upper panels show immunohistochemistry staining for αB‐crystallin in muscle fibres before exercise (A) and at 2 h after exercise (B). A fibre was considered positive if the staining inside the fibre was scattered and uneven (marked with red asterisks). Fibres were considered negative if the staining was homogeneous (all fibres in the left image). Lower panels show immunohistochemistry staining for myosin heavy chain IIA and IIX (SC71 antibody) in neighbouring sections. Before exercise, there was more αB‐crystallin protein present in type I fibres (marked ‘I’ in panel B, C and D), whereas after exercise, the scattered αB‐crystallin staining was found mainly in type II fibres (panel D). Scale bar represents 100 μm. n = 9. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 10. Post‐exercise changes in serum creatine kinase activity

Data are presented as the geometric mean ± 95% confidence interval. n = 9. ^* P < 0.05 versus pre‐exercise value.