Novel methods for cold exposure of skeletal muscle in vivo and in vitro show temperature-dependent myokine production.

Proteins secreted from skeletal muscle serving a signalling role have been termed myokines. Many of the myokines are exercise factors, produced and released in response to muscle activity. Cold exposures affecting muscle may occur in recreational, occupational and therapeutic settings. Whether muscle temperature independently affects myokine profile, is still to be elucidated. We hypothesized that manipulating muscle temperature by means of external cooling would change myokine production and release. In the present study we have established new models for cold exposure of muscle in vivo and in vitro where rat hind limb or cultured human myotubes were cooled to 18 °C. After a recovery period, muscle tissue, cells and culture media were harvested for further analysis by qPCR and immunoassays. Expression of several myokine genes were significantly increased after cold exposure in both models: in rat muscle, mRNA levels of CCL2 (p = 0.04), VEGFA (p = 0.02), CXCL1 (p = 0.02) and RBM3 (p = 0.02) increased while mRNA levels of IL-6 (p = 0.03) were decreased; in human myotubes, mRNA levels of IL6 (p = 0.01), CXCL8 (p = 0.04), VEGFA (p = 0.03) and CXCL1 (p < 0.01) were significantly increased, as well as intracellular protein levels of IL-8 (CXCL8 gene product; p < 0.01). The corresponding effect on myokine secretion was not observed, on the contrary, IL-8 (p = 0.02) and VEGF (VEGFA gene product) p < 0.01) concentrations in culture media were reduced after cold exposure in vitro. In conclusion, cold exposure of muscle in vivo and in vitro had an effect on the production and release of several known exercise-related myokines. Myokine expression at the level of mRNA and protein was increased by cold exposure, whereas secretion tended to be decreased.

SARS-CoV-2 serological findings and exposure risk among employees in school and retail after first and second wave COVID-19 pandemic in Oslo, Norway: a cohort study.

OBJECTIVES: The objective was to characterize and compare SARS-CoV-2 serology among Norwegian school employees and retail employees, and describe preventive measures taken at the workplaces. MATERIAL AND METHODS: A cohort of 238 school and retail employees was enrolled to an ambidirectional cohort study after the first COVID-19 pandemic wave. Self-reported exposure history and serum samples were collected at 10 schools and 15 retail stores in Oslo, Norway, sampled at 2 time-points: baseline (May-July 2020); and follow-up (January-March 2021). SARS-CoV-2 antibodies targeting both spike and nucleocapsid were detected by multiplex microsphere-based serological methods. RESULTS: At baseline, 6 enrolled workers (5 in retail) presented with positive SARS-CoV-2 serology, higher than the expected 1% prevalence (3%, 95% CI: 1-6, p = 0.019). At followup, school and retail groups presented 11 new seropositive cases altogether, but groups were not significantly different, although exposure and preventive measures against viral transmission at workplaces were different between groups. Self-reported medical history of COVID-19 infection showed that all but one positive SARS-CoV-2 serological findings arising between baseline and follow-up had been diagnosed with virus testing. CONCLUSIONS: Distribution of SARS-CoV-2 positive serology after the first wave was slightly higher than expected. Distribution of infection was not significantly different between the groups at baseline nor at follow-up, despite difference in exposure and protective measures. Nearly all new seropositive cases discovered between baseline and follow-up, had already been diagnosed, highlighting the importance of extensive viral testing among workers. Int J Occup Med Environ Health. 2022;35(5):537-47.

Experimental Models for Cold Exposure of Muscle in vitroand in vivo.

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.

Epiregulin is released from intervertebral disks and induces spontaneous activity in pain pathways.

INTRODUCTION: Lumbar radicular pain after disk herniation is associated with local release of many inflammatory molecules from nucleus pulposus (NP) cells leaking out of the intervertebral disk. Here, we have used a rat model to investigate the role of epiregulin (EREG), a member of the epidermal growth factor (EGF) family, in this process. METHODS: A protein immunoassay was chosen to confirm the release of EREG from the NP tissue. Single unit recordings were used to demonstrate the effect of recombinant EREG applied onto the dorsal nerve roots in vivo. Intracellular responses induced by recombinant EREG were studied in cultured dorsal root ganglion (DRG) cells by phosphoprotein assay. Changes in EGF receptor expression induced by NP in the DRG were examined by quantitative polymerase chain reaction. RESULTS: The protein immunoassay showed that EREG was released from the NP tissue. Moreover, application of EREG onto the spinal dorsal nerve roots induced a decrease in the evoked responses, but an increase in spontaneous activity in the dorsal horn neurons. Interestingly, the EREG activated the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in the DRG, a pathway previously linked to cellular growth, proliferation, and tissue regeneration. An NP-induced upregulation of the EGF receptor HER3 in the DRG was also revealed. CONCLUSION: Taken together, the present observations indicate that EREG may induce changes in the DRG and spontaneous activity in the pain pathways. We suggest that EREG signaling may be involved in the pathophysiological process leading to sensory deficits and neuropathic pain in patients after disk herniation.

Up-regulation of circulating microRNA-17 is associated with lumbar radicular pain following disc herniation.

BACKGROUND: Previous studies suggest that regulatory microRNAs (miRs) may modulate neuro-inflammatory processes. The purpose of the present study was to examine the role of miR-17 following intervertebral disc herniation. METHODS: In a cohort of 97 patients with leg pain and disc herniation verified on MRI, we investigated the association between circulating miR-17 and leg pain intensity. A rat model was used to examine possible changes in miR-17 expression in nucleus pulposus (NP) associated with leak of NP tissue out of the herniated disc. The functional role of miR-17 was addressed by transfection of miR-17 into THP-1 cells (human monocyte cell line). RESULTS: An association between the level of miR-17 in serum and the intensity of lumbar radicular pain was shown. Up-regulation of miR-17 in the rat NP tissue when applied onto spinal nerve roots and increased release of TNF following transfection of miR-17 into THP-1 cells were also observed. Hence, our data suggest that miR-17 may be involved in the pathophysiology underlying lumbar radicular pain after disc herniation. CONCLUSIONS: We conclude that miR-17 may be associated with the intensity of lumbar radicular pain after disc herniation, possibly through a TNF-driven pro-inflammatory mechanism.