Inflammation Is a Histological Characteristic of Skeletal Muscle in Chronic Limb Threatening Ischemia.

ABSTRACT

BACKGROUND: The loss of skeletal muscle is a prognostic factor in several diseases including in patients with chronic limb threatening ischemia (CLTI). Patients with CLTI also have a lower skeletal mass and area when compared to those with claudication. However, there are no currently available data regarding the histological characteristics of core muscles in patients with CLTI. This study aims to determine the differences in core skeletal muscles between patients with claudication and those with CLTI. The second aim is to evaluate the differences in myokines, which are molecules secreted by skeletal muscle, between patients with claudication and those with CLTI. METHODS: An observational, prospective study was conducted from January 2018 to July 2022 involving consecutive patients with peripheral arterial disease (PAD). The clinical characteristics were registered. In PAD patients with surgical indication for common femoral artery approach, samples of sartorius skeletal muscle (and not from the limb muscles directly involved in the ischemic process) were collected. The samples were submitted to histological characterization on hematoxylin-eosin and to immunohistochemical analysis to detect CD45+ leukocytes and CD163+ macrophages. The extent of the inflammatory cells (leukocytes and macrophages) was semiquantitatively assessed using a 0-to-4 grade scale as follows absent (0†), mild (†), moderate (††), severe (†††), and very severe (††††). Serum levels of myokines irisin, myostatin, IL-8, and lL-6 were determined with multiplex bead-based immunoassay. RESULTS: 119 patients (mean age 67.58 ± 9.60 years old, 79.80% males) 64 with claudication and 54 with CLTI were enrolled in the study. No differences were registered between patients with claudication and those with CLTI on age, gender, cardiovascular risk factors, and medication, except on smoking habits. There was a significantly higher prevalence of smokers and a higher smoking load in the claudication group. Samples of sartorius skeletal muscle from 40 patients (14 with claudication and 26 with CLTI) were submitted to histological analysis. No differences were found in skeletal muscle fibers preservation, trauma, or hemorrhage (on hematoxylin-eosin staining). However, in the immunohistochemistry study, we found more inflammatory cells CD45+ leukocytes in patients with CLTI when compared to those with claudication [CD45+ ≥ moderate (††) claudication (n = 14) 4; 28.57%; CLTI (n = 25) 16; 64.00%; P = 0.034]. Patients with CLTI also had higher tissue levels of CD163+ macrophages, but this difference was not significant [CD163+ ≥ moderate (††) claudication (n = 13) 7; 53.85%; CLTI (n = 27) 21; 77.78%; P = 0.122]. The serum levels of the myokines, irisin, and myostatin were below the lower limit of detection, in the majority of patients, so no valid results were obtained. However, patients with CLTI had a higher serum level of Interleukin (IL)-6 and IL-8. CONCLUSIONS: CLTI patients exhibit increased quantities of leukocytes in their sartorius muscle, as well as elevated serum levels of myokines IL-8 and IL-6. Inflamed skeletal muscle can contribute to the loss of muscle mass and account for the lower density of skeletal muscle observed in CLTI. Additionally, inflamed skeletal muscle may contribute to the development of systemic inflammation through the secretion of pro-inflammatory cytokines into the systemic circulation. Halting the inflammatory process could eventually improve the prognosis of CLTI patients.