Morphological Characterisation of Nerves in Whole Mount Airway Biopsies

Rationale: Neuroplasticity of bronchopulmonary afferent neurons that respond to mechanical and chemical stimuli may sensitize the cough reflex. Afferent drive in cough is carried by the vagus nerve and vagal afferent nerve terminals have been well defined in animals. Yet, both unmyelinated C-fibres and particularly the morphologically distinct, myelinated, nodose derived, mechanoreceptors described in animals are poorly characterized in humans. To date there are no distinctive molecular markers nor detailed morphologies available for human bronchopulmonary afferent nerves. Objective: Morphological and neuromolecular characterization of the afferent nerves that are potentially involved in cough in humans Methods: A whole mount immunofluorescence approach, rarely employed in human lung tissue, was used, utilizing antibodies specific to protein gene product 9.5 (PGP9.5) and, for the first time in human lung tissue, 200kDa neurofilament subunit. Results: We have developed a robust technique to visualize fibres consistent with autonomic and C-fibres as well as pulmonary neuroendocrine cells. A group of morphologically distinct, 200kDa neurofilament immunopositive myelinated afferent fibres, a sub-population of which did not express PGP9.5 were also identified. Conclusion: PGP9.5 immuno-negative nerves are strikingly similar to myelinated airway afferents; the cough receptor and smooth muscle associated airway receptors described in rodents. These have never before been described in humans. Full description of human airway nerves is critical to the translation of animal studies to the clinical setting.