Combined laryngeal inflammation and trauma mediate long-lasting immunoreactivity response in the brainstem sensory nuclei in the rat

Abstract

Somatosensory feedback from the larynx plays a critical role in regulation of normal upper airway functions, such as breathing, deglutition, and voice production, while altered laryngeal sensory feedback is known to elicit a variety of pathological reflex responses, including persistent coughing, dysphonia, and laryngospasm. Despite its clinical impact, the central mechanisms underlying the development of pathological laryngeal responses remain poorly understood. We examined the effects of persistent vocal fold (VF) inflammation and trauma, as frequent causes of long-lasting modulation of laryngeal sensory feedback, on brainstem immunoreactivity in the rat. Combined VF inflammation and trauma were induced by injection of lipopolysaccharide (LPS) solution and compared to VF trauma alone from injection of vehicle solution and to controls without any VF manipulations. Using a c-fos marker, we found significantly increased Fos-like immunoreactivity (FLI) in the bilateral intermediate/parvicellular reticular formation (IRF/PCRF) with a trend in the left solitary tract nucleus (NTS) only in animals with combined LPS-induced VF inflammation and trauma. Further, FLI in the right NTS was significantly correlated with the severity of LPS-induced VF changes. However, increased brainstem FLI response was not associated with FLI changes in the first-order neurons of the laryngeal afferents located in the nodose and jugular ganglia in either group. Our data indicate that complex VF alterations (i.e., inflammation/trauma vs. trauma alone) may cause prolonged excitability of the brainstem nuclei receiving a direct sensory input from the larynx, which, in turn, may lead to (mal)plastic changes within the laryngeal central sensory control.

Keywords: brainstem; immunoreactivity; inflammation; larynx; rat.

Figure 1

(A) Schematic drawing of the transverse section of the larynx in the rat, depicting the site of LPS or vehicle injection in the right vocal fold. (B) Photomicrograph of the right vocal fold depicts changes observed following 72 h after LPS injection; the magnified inset shows chronic inflammation and tissue re-organization at the site of LPS injection, characterized by the accumulation of macrophages and lymphocytes in the vocal fold mucosa, disintegrated muscle fibers, and increased vascularization and granulations. (C) Photomicrograph of the right vocal fold shows the site of vehicle injection; the magnified inset depicts the absence of formed inflammatory tissue response to trauma from the needle penetration as observed during LPS injection with only few scattered macrophages in the vocal fold tissue surrounding the needle tract. TA, thyroarytenoid muscle; AC, arytenoid cartilage; TC, thyroid cartilage; CC, cricoid cartilage.

Figure 2

Distribution of the Fos-positive neurons in the brainstem nuclei 72 h after LPS (A) and vehicle (B) injections into the right vocal fold and in anesthetic control without any laryngeal manipulations (C). Schematic diagrams depict increased FLI in a single representative animal per respective group. Corresponding photomicrographs below the diagrams show Fos activation in the right NTSi (arrowheads mark Fos-positive nuclei). DLPAG, dorsolateral nucleus of the PAG; DMPAG, dorsomedial nucleus of the PAG; LPAG, lateral nucleus of the PAG; VLPAG, ventrolateral nucleus of the PAG; IRF, intermediate reticular formation; LC, locus coeruleus; NA, nucleus ambiguus; NTS, solitary tract nucleus; NTSi, interstitial nucleus of the NTS; NTSim, intermediate nucleus of the NTS; NTSm, medial nucleus of the NTS; PAG, periaqueductal gray; PCRF, parvicellular reticular formation; sol, solitary tract; Sp5, spinal trigeminal nucleus. Diagrams were based on the atlas of the rat brain (Paxinos and Watson, 1998).

Figure 3

The bar graphs show the mean number of Fos-positive neurons in the examined brainstem nuclei in the LPS-, vehicle-treated and anesthetic controls. (A) Double asterisks (^**) mark significant FLI in the IRF/PCRF nucleus, while single asterisk (^*) marks a trend in FLI increase in the NTSi nucleus in LPS-treated animals compared to vehicle-treated animals and anesthetic controls. (B) No statistically significant differences between these groups were found within the higher-order nuclei of laryngeal afferents (PAG) and non-specific nuclei of laryngeal control (LC and AP). IRF/PCRF, intermediate/parvicellular reticular formation; NTS, solitary tract nucleus; Sp5, spinal trigeminal nucleus; PAG, periaqueductal gray; LC, locus coeruleus; AP, area postrema; NTSi, interstitial nucleus of the NTS; NTSim, intermediate nucleus of the NTS; NTSm, medial nucleus of the NTS; L, left; R, right.

Figure 4

Significant positive relationship between the mean number of Fos-positive neurons in the right NTSi and the extent of VF changes (in %) following LPS-induced inflammation and trauma.

Figure 5

Photomicrographs of the nodose ganglion in the iSLN-stimulated (A) and LPS-treated (B) animals. The arrows mark Fos-positive neurons in the iSLN-stimulated animal. No FLI increase was observed in any LPS- or vehicle-treated animal. GN, ganglion nodosum.