Diacetyl increases sensory innervation and substance P production in rat trachea

Abstract

Inhalation of diacetyl, a butter flavoring, causes airway responses potentially mediated by sensory nerves. This study examines diacetyl-induced changes in sensory nerves of tracheal epithelium. Rats (n = 6/group) inhaled 0-, 25-, 249-, or 346-ppm diacetyl for 6 hr. Tracheas and vagal ganglia were removed 1-day postexposure and labeled for substance P (SP) or protein gene product 9.5 (PGP9.5). Vagal ganglia neurons projecting to airway epithelium were identified by axonal transport of fluorescent microspheres intratracheally instilled 14 days before diacetyl inhalation. End points were SP and PGP9.5 nerve fiber density (NFD) in tracheal epithelium and SP-positive neurons projecting to the trachea. PGP9.5-immunoreactive NFD decreased in foci with denuded epithelium, suggesting loss of airway sensory innervation. However, in the intact epithelium adjacent to denuded foci, SP-immunoreactive NFD increased from 0.01 ± 0.002 in controls to 0.05 ± 0.01 after exposure to 346-ppm diacetyl. In vagal ganglia, SP-positive airway neurons increased from 3.3 ± 3.0% in controls to 25.5 ± 6.6% after inhaling 346-ppm diacetyl. Thus, diacetyl inhalation increases SP levels in sensory nerves of airway epithelium. Because SP release in airways promotes inflammation and activation of sensory nerves mediates reflexes, neural changes may contribute to flavorings-related lung disease pathogenesis.

Keywords: airway epithelium; airway inflammation; airway innervation; cough; flavorings; inhalation toxicity.

Figure 1

Classification of histopathologic alterations in respiratory epithelium. Images are from tracheas of rats inhaling 356 ppm in a previous study (Hubbs et al. 2008) and the rat nose from a high-dose animal in this study. (A) Intact respiratory epithelium in a diacetyl-exposed rat trachea. (B) Attenuated epithelium (solid arrow) in the diacetyl-exposed trachea may represent an attempt at sliding repair of denuded epithelium (dashed arrow). (C) Tracheal epithelial necrosis with detachment. (D) Respiratory epithelial detachment (solid arrow) and denuded basement membrane (dashed arrow) in the nose of a rat in the high-dose group from the current study. Hematoxylin and eosin stain; bar = 20 μm.

Figure 2

Immunofluorescence staining for SP (green) and red fluorescent microspheres in nerve cell bodies of jugular ganglion using rabbit anti-SP primary and fluorescein-labeled goat anti-rabbit secondary. (A) SP immunoreactivity in neurons of a jugular ganglion. (B) Red fluorescent microspheres in cell bodies of jugular ganglion from A. The microspheres indicate cell bodies of neurons projecting to the airway epithelium. (C) Merged images from A and B with SP-positive airway neurons identified by fluorescent microspheres. Solid arrows indicate two SP-positive airway neurons. Dashed arrow indicates airway neuron lacking SP immunoreactivity. SP = substance P.

Figure 3

Percentage of SP-positive airway neurons in nodose–jugular ganglia of rats 18 to 20 hr after air or 25.5-, 249-, or 346-ppm diacetyl exposure. SP = substance P. *p ≤ .05.

Figure 4

SP-immunoreactive nerves (arrows) in intact or damaged regions of tracheal epithelium (E): (A) SP-IR nerves in the intact epithelium. (B) SP-positive nerves in epithelium adjacent to denuded regions after 346-ppm diacetyl. (C) SP innervation in region of detaching epithelium. (D) SP nerves in attenuated epithelium. (E) Lack of SP-immunoreactive nerves in regions where epithelium was denuded. A few positive nerves are located below the basement membrane (arrow). SP = substance P; SP-IR = SP immunoreactive.

Figure 5

SP and PGP9.5 nerve fiber density in five different regions of epithelial damage. (A) SP is increased in the epithelium adjacent to denuded epithelium. In the highest exposure group, SP is also increased in the detaching epithelium. Air and 25-ppm exposure contained only intact epithelium. Insufficient data for significance of detaching epithelium in 249-ppm group. (B) PGP9.5 nerve fiber density. Insufficient data for attenuated epithelium in 249-ppm group. SP = substance P; PGP9.5 = protein gene product 9.5. *Significantly greater than the control. ^§Significantly less than the control. p ≤ .05.