The pulmonary neuroepithelial body-vagal sensory nerve axis is altered in a mouse model of bronchopulmonary dysplasia (BPD)

Abstract

Breathing control problems, like apnea of prematurity, are a common complication in preterm infants with bronchopulmonary dysplasia (BPD), but the underlying factors remain incompletely understood. A subpopulation of pulmonary vagal afferent nerve fibers (Calb1+) innervate CO2 and O2 sensitive clusters of lung neuroendocrine cells (neuroepithelial bodies (NEBs)) located along conducting airways. Calb1+ fibers provide direct sensory input to the brainstem that putatively limit tidal volume. NEBs are hyperplastic in BPD infants and may increase activity of inhibitory Calb1+ vagal fibers. Herein, we sought to determine if the NEB-Calb1 lung-brainstem axis is altered in association with breathing control in BPD. BPD in mice was established by exposure to chronic neonatal (postnatal day (P) 0-14) hyperoxia (Hx; 70% O2; n=20) or normoxia (Nx; n=17). Consistent with human BPD, Hx caused alveolar simplification supported by a reduction in alveolar density and alveoli (p<0.05; separate t-tests) in P22 H&E-stained lung sections (n=5-7/group). Like human infants at risk for BPD, Hx mice adapted to increased dead space (caused by reduction in alveoli) by hyperventilating (p<0.05; generalized linear modeling accounting for fat free mass and heat production, measured by NMR and respirometry; n=10-24/group). This was facilitated by an increase in tidal volume that was significantly reduced at P14 but increased at P17 and P21 (n=19-28) (Tukey’s post-hoc: p<0.05). NEBs and associated innervation (vagal and dorsal root ganglia) were next assessed in subsets of mice. First, IT instillation of WGA-594 lectin into subsets of mice (n=9 mice/18 ganglia per group) and subsequent quantification of 594+ neurons per vagal ganglia revealed fewer neurons (p=0.07; t-test) in Hx mice (110.8 ± 6.9 vs 95.2 ± 4.9). Second, CGRP+ innervation to NEBs is reduced in Hx mice (Quantification of CGRP+ nerve fibers only: 17740.4 +/- 4126 vs 10474.6 +/- 2140 +CGRP pixels; p=0.12 n=5-7/group). Third, there was greater frequency of NEBs per unit airway (p<0.05; 0.306 ± 0.03 vs 0.401 ± 0.03) with a tendency for larger NEBs (p=0.06; 9.14 ± 0.7 vs 11.93 ± 1.3) indicating NEB hyperplasia. Together, these data indicate individual NEB innervation is reduced in BPD but that total NEB innervation may remain unchanged. If this is true for Calb1+ vagal fibers remain untested. To functionally assess Calb1-vagal fibers we activated, with DCZ, the excitatory synthetic Gq-DREAD receptor (DTR) expressed in Calb1cre mice via microinjection of AAV-Cre-Gq-mCherry into the left vagal ganglia. DCZ but not vehicle caused a major increase in peak inspiratory flow during acute hypoxic challenges compared to vehicle treated mice (Pre-DMSO, Post-DMSO, Pre-DCZ = ~1ml/sec vs 4ml/sec post-DCZ; p<0.05, n=4/group). Collection of vagal ganglia confirms DTR expression but spillover of DTR is present. Repeating these studies with diluted AAV-Cre-Gq in control and BPD mice are ongoing. Together, our data verify that hyperoxia induced BPD in mice causes a similar reduction in alveoli and compensation to increased dead space by hyperventilation as observed in human BPD. Additionally, BPD structurally alters the NEB-vagal axis but whether these changes are functionally significant to nCOB is being addressed with ongoing studies. AHA 20CDA35310121, Advancing Healthier Wisconsin Endowment. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.