Structural and Functional Vocal Fold Epithelial Integrity Following Injury

Objectives/Hypothesis: An intact epithelium is an important part of vocal fold defense. Damage to the epithelium can compromise vocal fold homeostasis and protection of the host tissue from viral and bacterial invasion. Elucidating the effects of damage on epithelial architectural and barrier integrity provides insight into the role of epithelium in protecting vocal folds. Using an animal model, we evaluated the time course of structural and functional epithelial restoration following injury. Study Design: Prospective, controlled animal study. Methods: Forty rats underwent surgery to remove vocal fold mucosa unilaterally. Larynges were harvested at five time intervals between 3 to 90 days postinjury and were prepared for histological and permeability analyses. Results: Rapid restoration of structural integrity was demonstrated by return of a multilayerd epithelium, intercellular junctions, and basement membrane at 5 days postinjury. Atypical epithelial permeability was observed up to 5 weeks postinjury. Conclusion: Restoration of epithelial barrier integrity lags epithelial structural restoration. Consequently, epithelial regeneration cannot be equated with return of functional barrier integrity. Rather, ongoing leakiness of regenerated epithelium indicates that vocal folds remain at risk for damage, pathogen invasion, and remodeling postinjury

Classification for Animal Vocal Fold Surgery: Resection Margins Impact Histological Outcomes of Vocal Fold Injury

Objective—Extent of vocal fold injury impacts the nature and timing of wound healing, and voice outcomes. However, depth and extent of the lesion created to study wound healing in animal models vary across studies, likely contributing to different outcomes. Our goal was to create a surgery classification system to enable comparison of postoperative outcomes across animal vocal fold wound healing studies. Study design—Prospective, controlled animal study. Methods—Rats underwent one of three types of unilateral vocal fold surgeries classified by depth and length of resection. The surgeries were a subepithelial injury, resection of epithelium and superficial layer of the lamina propria at the midmembranous portion of the vocal fold; transmucosal injury, resection of epithelium and lamina propria; and transmuscular injury, resection of epithelium, lamina propria and superficial portion of the vocalis muscle Wound healing was evaluated histologically at various time points up to 35 days post-injury. Results—Complete healing occurred by 14 days post-surgery for subepithelial injury and by day 35 for transmucosal injury. Injury remained present at day 35 for transmuscular injury. Conclusions—Timing and completeness of healing varied by extent and depth of resection. Scarless healing occurred rapidly following subepithelial injury, while scarring was observed at five weeks after transmuscular injury. The proposed classification system may facilitate comparison of surgical outcomes across vocal fold wound healing studies

Flowchart for selecting an appropriate surgical airway in neurologically impaired pediatric intubated patients: a case series

Objective: Medical advances have resulted in increased survival rates of neurologically impaired children who may require mechanical ventilation and subsequent tracheostomy as a surgical airway. However, at present, there is no definite consensus regarding the timing and methods for placement of a surgical airway in a neurologically impaired intubated child who needs to be cared for over a long-term period. We therefore created a flowchart for the selection of a surgical airway for Neurologically Impaired Pediatric Patients (NIPPs). Methods: The flowchart includes information on the patients’ backgrounds, such as intubation period, prognosis related to reversibility, and history of aspiration pneumonia. To evaluate the importance of the flowchart, first we conducted a survey of pediatricians regarding selection of a surgical airway, and we also evaluated the appropriateness of the flowchart among pediatricians and caregivers through questionnaire surveys which include satisfaction with the decision-making process, and postoperative course after discharge. Results: A total of 21 NIPPs with intubation underwent surgery and a total of 24 participants (14 pediatricians and 10 caregivers) completed the survey. The answers regarding the importance of the flowchart showed that eleven pediatricians had experience selecting of surgical airways, nine of whom had had experiences in which they had to make a difficult decision. The answers regarding the appropriateness of the flowchart revealed that all pediatricians and caregivers were satisfied with the decision-making process and postoperative course after discharge using the flowchart. Conclusions: The present study demonstrated the effectiveness of our flowchart for selecting an appropriate surgical airway in NIPP. By referring to our flowchart, pediatricians and caregivers are likely to be able to select an appropriate surgical airway, leading to increased satisfaction with the decision-making process and postoperative course. Level of Evidence: 4

Epithelial Cells Are Active Participants in Vocal Fold Wound Healing: An In Vivo Animal Model of Injury

Vocal fold epithelial cells likely play an important, yet currently poorly defined, role in healing following injury, irritation and inflammation. In the present study, we sought to identify a possible role for growth factors, epidermal growth factor (EGF) and transforming growth factor-beta 1 (TGFβ1), in epithelial regeneration during wound healing as a necessary first step for uncovering potential signaling mechanisms of vocal fold wound repair and remodeling. Using a rat model, we created unilateral vocal fold injuries and examined the timeline for epithelial healing and regeneration during early and late stages of wound healing using immunohistochemistry (IHC). We observed time-dependent secretion of the proliferation marker, ki67, growth factors EGF and TGFβ1, as well as activation of the EGF receptor (EGFR), in regenerating epithelium during the acute phase of injury. Ki67, growth factor, and EGFR expression peaked at day 3 post-injury. Presence of cytoplasmic and intercellular EGF and TGFβ1 staining occurred up to 5 days post-injury, consistent with a role for epithelial cells in synthesizing and secreting these growth factors. To confirm that epithelial cells contributed to the cytokine secretion, we examined epithelial cell growth factor secretion in vitro using polymerase chain reaction (PCR). Cultured pig vocal fold epithelial cells expressed both EGF and TGFβ1. Our in vivo and in vitro findings indicate that epithelial cells are active participants in the wound healing process. The exact mechanisms underlying their roles in autocrine and paracrine signaling guiding wound healing await study in a controlled, in vitro environment

Epithelial Cells Are Active Participants in Vocal Fold Wound Healing: An <i>In Vivo</i> Animal Model of Injury

<div><p>Vocal fold epithelial cells likely play an important, yet currently poorly defined, role in healing following injury, irritation and inflammation. In the present study, we sought to identify a possible role for growth factors, epidermal growth factor (EGF) and transforming growth factor-beta 1 (TGFβ1), in epithelial regeneration during wound healing as a necessary first step for uncovering potential signaling mechanisms of vocal fold wound repair and remodeling. Using a rat model, we created unilateral vocal fold injuries and examined the timeline for epithelial healing and regeneration during early and late stages of wound healing using immunohistochemistry (IHC). We observed time-dependent secretion of the proliferation marker, ki67, growth factors EGF and TGFβ1, as well as activation of the EGF receptor (EGFR), in regenerating epithelium during the acute phase of injury. Ki67, growth factor, and EGFR expression peaked at day 3 post-injury. Presence of cytoplasmic and intercellular EGF and TGFβ1 staining occurred up to 5 days post-injury, consistent with a role for epithelial cells in synthesizing and secreting these growth factors. To confirm that epithelial cells contributed to the cytokine secretion, we examined epithelial cell growth factor secretion <i>in vitro</i> using polymerase chain reaction (PCR). Cultured pig vocal fold epithelial cells expressed both EGF and TGFβ1. Our <i>in vivo</i> and <i>in vitro</i> findings indicate that epithelial cells are active participants in the wound healing process. The exact mechanisms underlying their roles in autocrine and paracrine signaling guiding wound healing await study in a controlled, in vitro environment.</p></div

Growth factor expression in primary pig vocal fold epithelial cells.

<p>Agarose gel electrophoresis of PCR products shows expression of EGF (lane one), TGFβ1 (lane two), and the positive control (lane five) in primary pig vocal fold epithelial cells at the expected product sizes. Negative water controls (W) for EGF and TGFβ1 are shown in lanes three and four, respectively.</p

Epithelial regeneration following vocal fold injury.

<p>Hematoxylin and eosin (H&E) in injured vocal folds from day 1 (D1) to day 35 (D35) post-injury and uninjured vocal folds (A–F). Immunostaining (brown) for keratin 14 (K14; G–L); Ki67 (M–R); epidermal growth factor (S–X); and transforming growth factor beta (TGFβ1) at five time points ranging from 1 to 35 days post-injury, as well as in uninjured vocal folds (Y-DD). Images show results for representative animals. Scale bar: 100 µm.</p

Primer sequences and products for polymerase chain reaction (PCR).

<p>Primer sequences and products for polymerase chain reaction (PCR).</p