The NEB-P2ry1 lung-brainstem axis and the control of breathing in a mouse model of bronchopulmonary dysplasia
Authors: Mouradian Gary , Cook-Snyder Denise , Dir Brooke , Segar Jeffrey , Hodges Matthew , Grobe Justin 
Published: May 2023

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 (P2ry1+) innervate CO2 and O2 sensitive clusters of lung neuroendocrine cells (neuroepithelial bodies (NEBs)) located along conducting airways. P2ry1 fibers provide direct sensory input to the brainstem and inhibit breathing upon stimulation. NEBs are hyperplastic in BPD infants and may increase activity of inhibitory P2ry1 vagal fibers. Herein, we sought to determine if the NEB-P2ry1 lung-brainstem axis is altered in association with breathing control in BPD. BPD in mice was induced 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 (p<0.05) and alveoli (p<0.05) in P22 H&E-stained lung sections. Hx mice spent more time apneic at P14 and P17 (p<0.05) in room air and acute hypoxia. Overall (main effect), room air minute ventilation (VE) was reduced in HX mice (p<0.05). Further, VE was reduced during acute hypoxia at P14 and P17 (not P21) with an overall reduction of breathing in Hx mice (p<0.05). From a subset of mice (n=4-5/group) VCO2, VO2, and heat production at rest were greater in P21 Hx mice during room air, suggesting the Hx mice were hypoventilating. Immunofluorescent staining of CGRP (hallmark NEB marker), Ki67 (proliferation), and SEZ6L2 (new NEB cell-membrane marker) in Hx and Nx lungs were imaged and analyzed by a blinded investigator. Consistent with clinical findings, Hx mice had hyperplastic NEBs (8.6 ± 0.45 vs 10.7 ± 1.3 cells/NEB; p<0.05) while only 2 of 143 NEBs expressed Ki67, suggesting no proliferation at P22. CGRP co-localized with SEZ6L2 in both Nx and Hx lungs. A subset of mice received intratracheal delivery of 1ug SEZ6L2-Saporin toxin to lesion NEBs and assess their function. 4 days after SEZ-Saporin treatment breathing frequency was elevated by 11% ± 5% in room air and by 7% ± 5% during acute hypoxia but not in PBS-treated, adult mice (n=3/group) suggesting NEBs have a mild inhibitory influence under normal conditions. Lastly, wireless optogenetic stimulation of P2ry1 vagal fibers in unanesthetized adult control mice (n=3) increased room air breathing by 20% ± 1% and drive to breathe by 36% ± 19% compared to pre-stim breathing. Together, our data demonstrate that hyperoxia-induced BPD alters the control of breathing which may in part be mediated via a unique lung-brainstem axis comprised of hyperplastic NEBs causing greater inhibition of excitatory P2ry1 vagal fibers. The NEB-P2ry1 axis may serve as a novel target for improving breathing in BPD. GCM: Advancing Healthier Wisconsin REAC Award, AHA 20CDA35310121; MRH: HL122358; JLG: DK133121, HL134850, and HL084207; JLS: DK133121 This is the full abstract presented at the American Physiology Summit 2023 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.