Parainfluenza 3-Induced Cough Hypersensitivity in the Guinea Pig Airways

<div><p>The effect of respiratory tract viral infection on evoked cough in guinea pigs was evaluated. Guinea pigs were inoculated intranasally with either parainfluenza type 3 (PIV3) and cough was quantified in conscious animals. The guinea pigs infected with PIV3 (day 4) coughed nearly three times more than those treated with the viral growth medium in response to capsaicin, citric acid, and bradykinin. Since capsaicin, citric acid, and bradykinin evoked coughing in guinea pigs can be inhibited by drugs that antagonize the transient receptor potential cation channel, subfamily V, member 1 (TRPV1), it was reasoned that the virally-induced hypertussive state may involve alterations in TPRV1 activity. PIV3 infection caused a phenotypic switch in tracheal nodose Aδ “cough receptors” such that nearly 50% of neurons began to express, de novo, TRPV1 mRNA. There was also an increase TRPV1 expression in jugular C-fiber neurons as determined by qPCR. It has previously been reported that tracheal-specific nodose neurons express the BDNF receptor TrkB and jugular neurons express the NGF receptor TrkA. Jugular neurons also express the artemin receptor GFRα3. All these neurotrophic factors have been associated with increases in TRPV1 expression. In an ex vivo perfused guinea pig tracheal preparation, we demonstrated that within 8 h of PIV3 infusion there was no change in NGF mRNA expression, but there was nearly a 10-fold increase in BDNF mRNA in the tissue, and a small but significant elevation in the expression of artemin mRNA. In summary, PIV3 infection leads to elevations in TRPV1 expression in the two key cough evoking nerve subtypes in the guinea pig trachea, and this is associated with a hypertussive state with respect to various TRPV1 activating stimuli.</p></div

TRPV1 expression in the jugular ganglia in control animals compared to those inoculated with PIV3 (day 4).

<p>Increased expression of TRPV1 (qPCR) following PIV3 infection for 4 d. Control: n = 7, 14 ganglia; PIV3: n = 7, 14 ganglia. *Significantly different from control (p<0.05).</p

PIV3 infection of epithelium in guinea pig trachea 4 d after inoculation with virus or vehicle (control).

<p>A) PIV3 staining of tracheal epithelium from a vehicle-treated control animal. B) PIV3 staining of tracheal epithelium following viral infection in vivo. Bar = 100 μm. C) Each lane represents RT-PCR amplification with primers for PIV3 genes: nucleocapsid protein (NP), fusion protein (FP), hemagglutinin-neuraminidase (HN), and a positive control- guinea pig β-actin (β).</p

Guinea pig cough responses to inhaled capsaicin (0.1, 1, 3, 10 μM), citiric acid (CA; 0.01, 0.1, 0.3 M) and bradykinin (BK; 0.1 mg/ml) 4 d after viral inoculation.

<p>Cough was confirmed in conscious guinea pigs based on changes in pressure and visual confirmation. Comparison of the average number of coughs between infected animals and controls were compared. A) Capsaicin: Control, n = 4; PIV3: n = 4. B) CA: Control, n = 8; PIV3, n = 8. D) Bradykinin: Control, n = 10; PIV3, n = 10. *Significantly different from control (p<0.05).</p

Sequence of primers used for PCR analysis of PIV3.

<p>Sequence of primers used for PCR analysis of PIV3.</p

Percentage of guinea pig tracheal-specific nodose neurons that express TRPV1 in controls compared to those inoculated with PIV3 (day 4).

<p>Top, representative mRNA expression of TRPV1 from neurons. Bottom, total TRPV1 mRNA expression in infected animals (49.48 ±5.1%) compared to controls (16.28 ±4.0%). Control: n = 11, 86 neurons; PIV3: n = 10, 97 neurons. *Significantly different from control (p<0.05).</p

Sequence of primers used for analysis of guinea pig ganglia receptor transcripts.

<p>Sequence of primers used for analysis of guinea pig ganglia receptor transcripts.</p

Sequence of primers used for qPCR analysis of guinea pig neurotrophin transcripts.

<p>Sequence of primers used for qPCR analysis of guinea pig neurotrophin transcripts.</p

Vehicle or viral infections of the trachea occurred ex vivo for qPCR analyses of neurotrophin levels.

<p>Upper left, a schematic of the isolated, perfused guinea pig airway apparatus used to virally infect intact epithelium. Lower right graph demonstrates an increased expression of BNDF and artemin (ARTN) in trachea following infection with 1/100 diluted PIV3 virus (stock = 2x10⁷ pfu/ml) for 8 h. The data are the average of 4 separate experiments. *Significantly different from control (p<0.05).</p