Characterization of the Leukocyte Response in Acute Vocal Fold Injury

Abstract

Macrophages location in the superficial layer of the vocal fold (VF) is not only at the first line of defense, but in a place of physiologic importance to voice quality. This study characterizes and compares macrophage function in two models of acute injury. Porcine VF injuries were created bilaterally by either surgical biopsy or lipopolysaccharide (LPS) (1.5μg/kg) injection. Animals were sacrificed at 1- or 5-day post LPS or 3-, 7-, or 23-days post-surgical injury (n = 3/time/ injury). Flow cytometry characterized immunophenotypes and RT-PCR quantified cytokine gene expression. Uninjured VF were used as controls. Post-surgical and LPS injury, SWC9+/SWC3- cells identified as hi SLA-DR+ (p<0.05) compared to controls along with hi CD16+ expression at 1-day and 3-days respectively compared to all other time points (p<0.05). Surgical injuries, SWC9+/SWC3- cells exhibited hi CD163+ (p<0.05) at 3-days along with upregulation in TNFα and TGFβ1 mRNA compared to 23-days (p<0.05). No measurable changes to IL–12, IFNγ, IL–10, IL–4 mRNA post-surgery. LPS injuries induced upregulation of TNFα, IL–12, IFNγ, IL–10, and IL–4 mRNA at 1- and 5-days compared to controls (p<0.05). Higher levels of IL–10 mRNA were found 1-day post-LPS compared to 5-days (p<0.05). No changes to CD163 or CD80/86 post-LPS were measured. Acute VF injuries revealed a paradigm of markers that appear to associate with each injury. LPS induced a regulatory phenotype indicated by prominent IL–10 mRNA expression. Surgical injury elicited a complex phenotype with early TNFα mRNA and CD163+ and persistent TGFβ1 transcript expression.

Acute exposure to vibration is an apoptosis‐inducing stimulus in the vocal fold epithelium

“…To confirm that cell death occurred, we found that the vocal fold epithelium expressed an immunohistochemical marker for apoptosis using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining (11). These findings are in agreement with the previously reported effects of mechanical trauma on cell death in vocal fold fibroblasts (12–14). Collectively, the literature suggests that apoptosis may be a natural biological response to vocal fold vibration and may therefore contribute to maintaining a normal epithelial barrier.…”

In vitro evaluation of anti‐fibrotic effects of select cytokines for vocal fold scar treatment

“…In order to establish cell phenotypes consistent with scar and short‐term biomaterial‐insertion, activation media containing LPS and TGF‐β1was used . We have previously demonstrated that this activation induces normal VFF to take on characteristics of a fibrotic phenotype, and AMO to take on phenotype similar to that seen in chronic scar as well as many biomaterial‐treated dermal wounds (loosely, a phenotype between the traditional pro‐ and anti‐inflammatory classifications).…”

“…Moreover, macrophages contribute to fibrosis through the production of many of these proteins, although the specific contribution of each is difficult to discern for reasons denoted in the “Introduction”. Previous studies related to characterizing macrophage phenotype (in response to cytokines or in vocal fold injuries) only examined two to three AMO markers, usually limited to a specific role (i.e. inflammation).…”

“…First, determining/understanding the underlying biological mechanisms is essential for the development of future strategies aimed at completely restoring vocal fold function. Second, injury to the vocal fold through cutting (as would be performed during surgery) has been shown to induce a significant activated macrophage (AMO)‐based inflammatory response, and macrophages have been shown to directly influence fVFF behavior …”

Alterations in macrophage polarization in injured murine vocal folds

“…Previous studies on normal human vocal folds have shown that tissue‐resident macrophages exist in the superficial layer of the lamina propria and respond to mucosal irritants in children and adults . Recent investigations have demonstrated that pan‐macrophage marker‐positive macrophages increased in the entire lamina propria from 12 hours through 7 days postinjury and return to baseline 2 to 4 weeks postinjury in injured rat vocal folds, and M2 marker‐positive macrophages increased in injured porcine vocal folds . However, the phenotypic or distributional changes in macrophages in the vocal fold during wound healing have not been well investigated.…”