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. 2017 Mar 7:8:93.
doi: 10.3389/fphys.2017.00093. eCollection 2017.

Effects of Topical Icing on Inflammation, Angiogenesis, Revascularization, and Myofiber Regeneration in Skeletal Muscle Following Contusion Injury

Daniel P Singh  1 , Zohreh Barani Lonbani  1 , Maria A Woodruff  2 , Tony J Parker  3 , Roland Steck  4 , Jonathan M Peake  3
Affiliations

Effects of Topical Icing on Inflammation, Angiogenesis, Revascularization, and Myofiber Regeneration in Skeletal Muscle Following Contusion Injury

Daniel P Singh et al. Front Physiol. .

Abstract

Contusion injuries in skeletal muscle commonly occur in contact sport and vehicular and industrial workplace accidents. Icing has traditionally been used to treat such injuries under the premise that it alleviates pain, reduces tissue metabolism, and modifies vascular responses to decrease swelling. Previous research has examined the effects of icing on inflammation and microcirculatory dynamics following muscle injury. However, whether icing influences angiogenesis, collateral vessel growth, or myofiber regeneration remains unknown. We compared the effects of icing vs. a sham treatment on the presence of neutrophils and macrophages; expression of CD34, von Willebrands factor (vWF), vascular endothelial growth factor (VEGF), and nestin; vessel volume; capillary density; and myofiber regeneration in skeletal after muscle contusion injury in rats. Muscle tissue was collected 1, 3, 7, and 28 d after injury. Compared with uninjured rats, muscles in rats that sustained the contusion injury exhibited major necrosis, inflammation, and increased expression of CD34, vWF, VEGF, and nestin. Compared with the sham treatment, icing attenuated and/or delayed neutrophil and macrophage infiltration; the expression of vWF, VEGF, and nestin; and the change in vessel volume within muscle in the first 7 d after injury (P < 0.05). By contrast, icing did not influence capillary density in muscle 28 d after injury (P = 0.59). The percentage of immature myofibers relative to the total number of fibers was greater in the icing group than in the sham group 28 d after injury (P = 0.026), but myofiber cross-sectional area did not differ between groups after 7 d (P = 0.35) and 28 d (P = 0.30). In conclusion, although icing disrupted inflammation and some aspects of angiogenesis/revascularization, these effects did not result in substantial differences in capillary density or muscle growth.

Keywords: angiogenesis; cryotherapy; inflammation; muscle injury; regeneration.

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Figures

Figure 1
Figure 1
Time course of experimental procedures. The uninjured control, icing, and sham treatment groups at each time of sacrifice were divided into groups of six rats for histology and immunohistochemistry, and four rats for micro-CT imaging.
Figure 2
Figure 2
Representative micro-CT image of the vascular network within a biopsy stack (250 slices) from one animal 1 d after injury.
Figure 3
Figure 3
Histogram showing the distribution of vessel size. Vessel size was determined by micro-CT (vessel diameter 6–78 μm) within a biopsy stack (250 slices) from one animal 1 d after injury.
Figure 4
Figure 4
Representative cross sections of skeletal muscle tissue. Muscle tissue was stained with H&E in the sham group (A–D) and the icing group (F–I) at 1, 3, 7, and 28 d after injury. (E) Shows muscle tissue from an uninjured rat for comparison. Arrowheads indicate necrotic muscle fibers. Arrows indicate regenerating muscle fibers (centrally placed nuclei). Scale bar = 100 μm.
Figure 5
Figure 5
Representative images of immunohistochemistry staining of neutrophils. Skeletal muscle tissue was stained with HIS48 antibody to identify neutrophils. Sham (A,C) and icing (B,D) groups at 1 and 3 d after injury. Scale bar = 20 μm.
Figure 6
Figure 6
Representative images of immunohistochemistry staining of macrophages. Skeletal muscle tissue was stained with CD68 antibody to identify macrophages. Sham (A–C) and icing (D–F) groups at 1, 3, and 7 d after injury. Arrows indicate macrophages. Scale bar = 20 μm.
Figure 7
Figure 7
Quantitative data for neutrophil (A) and macrophage (B) count in skeletal muscle tissue. n = 6 rats per group. Data are mean ± SD and are expressed as a fold-difference from uninjured control rats. Neutrophils and macrophages were not present in skeletal muscle tissue from uninjured control rats. Main effects for neutrophil counts: time (P < 0.001), group (P = 0.029), and group × time interaction (P < 0.001). Main effects for macrophage counts: time (P < 0.001), group (P = 0.008), and group × time interaction (P < 0.001). *P < 0.05 for unpaired t-test vs. uninjured control rats. #P < 0.05 for unpaired t-test vs. sham-treated rats.
Figure 8
Figure 8
Representative images of immunohistochemistry staining of endothelial cells. Skeletal muscle tissue was stained with CD34 antibody to identify endothelial cells. Sham (A–D) and icing (E–H) groups at 1, 3, 7, and 28 d after injury. Arrows indicate vessels. Scale bar = 50 μm.
Figure 9
Figure 9
Quantitative data for the area of positive staining for CD34 (A), vWF (B), VEGF (C), and nestin (D). The data are shown as a percentage relative to the total area of muscle tissue within the field of view. Data are mean ± SD, n = 6 rats per group, and are expressed as a fold difference relative to the uninjured control rats. Main effects for CD34: time effect (P < 0.001), group effect (P < 0.001), and group × time interaction (P < 0.001). Main effects for vWF: time effect (P < 0.001), group effect (P = 0.63), and group × time interaction (P < 0.001). Main effects for VEGF: time effect (P < 0.001), group effect (P = 0.56), and group × time interaction (P < 0.001). Main effects for nestin: time effect (P < 0.001), group effect (P = 0.98), and group × time interaction (P < 0.001). *P < 0.05 for unpaired t-test vs. uninjured control rats. #P < 0.05 for unpaired t-test vs. sham-treated rats.
Figure 10
Figure 10
Representative images of immunohistochemistry staining for vWF antibody to identify endothelial cells in muscle tissue. Sham (A–C) and icing (D–F) groups at 1, 3, 7, and 28 d after injury. Arrows indicate mature vessels. Scale bar = 50 μm.
Figure 11
Figure 11
Representative images of immunohistochemistry staining with VEGF antibody. Muscle tissue was obtained from the sham (A) and icing (B) groups at 3 d after injury. Arrows indicate vessels expressing VEGF. Arrowheads indicate positively stained macrophages. Scale bar = 50 μm.
Figure 12
Figure 12
Representative images of immunohistochemistry staining. Nestin antibody was used to identify endothelial cells in muscle tissue from the sham (A–D) and icing (E–H) groups at 1, 3, 7, and 28 d after injury. Arrows indicate positively stained endothelial cells. Scale bar = 50 μm.
Figure 13
Figure 13
Quantitative data for vessel volume (A) and number of regenerating myofibers (B) in skeletal muscle tissue. Data are mean ± SD. Data for vessel volume were obtained from n = 4 rats per group and are expressed as a fold difference relative to the uninjured control rats. Data for number of regenerating myofibers were obtained from n = 6 rats per group. Main effects for vessel volume: time effect (P = 0.098), group effect (P < 0.001), and group × time interaction (P < 0.001). #P < 0.05 for unpaired t-test vs. sham-treated rats.
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References

    1. Alva N., Azuara D., Palomeque J., Carbonell T. (2013a). Deep hypothermia protects against acute hypoxia in vivo in rats: a mechanism related to the attenuation of oxidative stress. Exp. Physiol. 98, 1115–1124. 10.1113/expphysiol.2012.071365 - DOI - PubMed
    1. Alva N., Palomeque J., Carbonell T. (2013b). Oxidative stress and antioxidant activity in hypothermia and rewarming: can RONS modulate the beneficial effects of therapeutic hypothermia? Oxid. Med. Cell. Longev. 2013:957054. 10.1155/2013/957054 - DOI - PMC - PubMed
    1. Amoh Y., Yang M., Li L., Reynoso J., Bouvet M., Moossa A. R., et al. . (2005). Nestin-linked green fluorescent protein transgenic nude mouse for imaging human tumor angiogenesis. Cancer Res. 65, 5352–5357. 10.1158/0008-5472.CAN-05-0821 - DOI - PubMed
    1. Birbrair A., Zhang T., Wang Z. M., Messi M. L., Olson J. D., Mintz A., et al. . (2014). Type-2 pericytes participate in normal and tumoral angiogenesis. Am. J. Physiol. Cell Physiol. 307, C25–38. 10.1152/ajpcell.00084.2014 - DOI - PMC - PubMed
    1. Canale S. T., Cantler E. D., Jr., Sisk T. D., Freeman B. L., III (1981). A chronicle of injuries of an American intercollegiate football team. Am. J. Sports Med. 9, 384–389. 10.1177/036354658100900608 - DOI - PubMed

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