Integrated Immunohistochemical and Ultrastructural Characterization of Layer-Specific Capillary Specialization in the Human Vocal Fold

The human true vocal fold exhibits a complex microvascular organization essential for its biomechanical and metabolic function. This study aimed to quantitatively assess CD31/PECAM-1-positive microvascular structures across the superficial lamina propria (SLP), deep lamina propria (DLP), and vocalis muscle (MV), and to integrate these findings with neuron-specific enolase (NSE) and scanning electron microscopy (SEM) observations. A retrospective analysis was performed on 21 formalin-fixed specimens. CD31 immunohistochemistry was used for endothelial identification, NSE immunohistochemistry was applied for the evaluation of neural elements, while SEM provided complementary ultrastructural information on extracellular matrix organization. Total microvascular density differed significantly among layers (χ2 = 32.12, df = 2, p = 1.06 × 10−7; Kendall’s W = 0.77), with highest values in MV (19.11 ± 6.22 vessels/field), followed by SLP (13.55 ± 3.93), and DLP (8.11 ± 2.41). Capillary density also showed significant inter-layer differences (p = 1.99 × 10−7), whereas small- and medium-caliber vessels did not (p = 0.081 and p = 0.538). NSE-positive neural profiles exhibited a similar distribution pattern, with higher density in the MV and lower values in the DLP. Inter-observer agreement was excellent (ICC = 0.91). Integrated analysis indicated a parallel spatial distribution of vascular, neural, and extracellular matrix components across vocal fold layers. This study provides a quantitative and structural baseline of the vocal fold microenvironment. This descriptive framework may inform future investigations of the layer-specific organization of vascular and neural-associated structures within the human vocal fold microenvironment.