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Time Course of Inflammatory Gene Expression Following Crush Injury in Murine Skeletal Muscle

Voss, Joachim G.; Shagal, Ayelet Goldshmid; Tsuji, Joyce M.; MacDonald, James W.; Bammler, Theo K.; Farin, Fred M.; St. Pierre Schneider, Barbara

doi: 10.1097/NNR.0000000000000209
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Background: Early inflammation and secretion of proinflammatory cytokines such as IL-1β, IL-6, and TNF-α act as the key drivers to regulate inflammation after muscle injury. However, the effects of these key proinflammatory drivers in a noninvasive crush injury model are not well known. Understanding these effects is important for treating crush injuries that occur during natural disasters and military conflicts.

Purpose: We studied the timed mRNA expression of IL-1β, IL-6, and TNF-α in a noninvasive murine crush injury model to further understand their impact on proinflammatory cytokine pathways that are activated within the first 48 hours after a crush muscle injury.

Methods: A total of 25 mice were anesthetized and placed on a crush injury apparatus platform with the apparatus piston situated in direct contact with intact skin overlying the right gastrocnemius muscle. Pressure at 45 psi was applied to the piston for 30 seconds for two applications. The mice recovered for either 4, 8, 24, or 48 hours postinjury, after which we harvested the gastrocnemius muscle of both legs. Microarray, confirmatory real-time polymerase chain reaction, and immunolabeling experiments were followed by a microarray time-course analysis.

Results: Muscle IL-1β mRNA rose 270-fold within 4 hours and declined rapidly at 8 hours to 196-fold, 24 hours to 96-fold, and 48 hours to 10-fold. Muscle IL-6 followed the same pattern, with a 34-fold increase at 4 hours, 29-fold increase at 8 hours, 10-fold increase at 24 hours, and 5-fold increase at 48 hours. Ingenuity Pathway Analysis of IL-6 identified activation of two major downstream signaling pathways (IL-6/Stat3 and IL-1β/Egr1) as key activators of inflammation, regeneration, and fibrosis.

Discussion: Closed crush muscle injury produced robust muscle cytokine expression levels, and the microarray findings allowed us to generate our most novel hypothesis: that high expression of IL-1β, IL-6, and TNF-α may be related to the downregulation of mitochondrial genes early after injury and triggers activation of genes in the repair and fibrosis machinery. The significance of these findings and the identified expression pathways of IL1-β, IL-6, and TNF-α and their downstream targets in skeletal muscle will allow us to further investigate targets for improved muscle recovery and limb-saving interventions.

Joachim G. Voss, PhD, RN, ACRN, FAAN, is Professor and Director, Sarah Cole Hirsh Institute for Evidence-Based Practice, Frances Payne Bolton School of Nursing, Case Western Reserve University, Cleveland, Ohio.

Ayelet Goldshmid Shagal, BS, MS, is PhD student, Department of Microbiology, Immunology & Genetics, Faculty of Health Sciences, Ben Gurion University, Beer-Sheva, Israel.

Joyce M. Tsuji, BS, MS, is Research Scientist, Department of Biological Structure, School of Medicine, University of Washington, Seattle.

James W. MacDonald, PhD, is Research Scientist, Environmental Health, University of Washington, Seattle.

Theo K. Bammler, PhD, is Research Scientist; and Fred M. Farin, MD, is Research Scientist, Environmental Health, University of Washington, Seattle.

Barbara St. Pierre Schneider, PhD, RN, CNE, is Research Professor, School of Nursing, University of Nevada, Las Vegas.

Accepted for publication September 25, 2016.

The authors acknowledge that this work was supported by the Air Force Surgeon General’s Office under Agreement Number FA7014-10-2-0001 (awarded to B. S.). The views expressed in this article are those of the authors and do not necessarily represent the official position or policy of the Air Force, the Department of Defense, or the U.S. Government.

The authors would like to thank Nadia D. Fulkerson, BS, MPH, for her technical assistance with the mouse experiments.

Editorial note: Dr. Kathleen Hickey was Action Editor for this paper.

The authors have no conflicts of interest to report.

Corresponding author: Joachim Voss, PhD, RN, ACRN, FAAN, Sarah Cole Hirsh Institute for Evidence-Based Practice, School of Nursing, Case Western Reserve University, 10099 Euclid Ave., Cleveland, OH 44106 (e-mail: joachim.voss@case.edu).

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