Unravelling lung interoception and its functional consequence in the developing ovine lung

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Project Summary The interoceptive link between the lung and CNS carries mechanical and irritant information to its first-order synapse in the brainstem. This information is critical in the newborn and adult for maintenance of pulmonary gas exchange in the face of behavioral changes and environmental stressors, and in orchestrating immune responses to viral, bacterial, and allergic pathogens. Although the fetal lung is richly innervated during development, the connectivity to the central nervous system (CNS), the extent to which these neurons help orchestrate lung and CNS formation over the course of development, and the role they play in function at birth remain largely unknown. This R01 application proposes to address this knowledge gap using a novel fetal sheep model. We hypothesize that intrinsic and extrinsic lung interoceptive units play a role in the normal development of the lung and central respiratory control centers. We will test this in the following aims: Aim 1: Traditional dye and novel viral track tracer techniques will be used in fetal sheep at various stages of lung development to delineate the location and connectivity of the interoceptive units within the lung, as well as first- and second-order central circuitry to and above the brainstem. The effect of surgical denervation on lung developmental morphology and the prevalence of various neural populations involved in chemo- and irritant sensation, including A- and C-fiber neurons involved in breathing control, and pulmonary neuroendocrine cells involved in pulmonary mechano- and chemosensation. Finally, single neuron RNAseq will be used to phenotypically characterize neurons within the fetal jugular and nodose ganglia at various stages of development. Aim 2: To test the necessity of interroception on fetal lung development and respiratory function after birth, fetuses in the canalicular stage will be subjected to either bilateral denervation of the lungs or periodic hyperstimulation of mechanoreceptors via overinflation for the remainder of gestation. Pulmonary vascular function, airway mechanics, and gas exchange will then be monitored following c-section delivery and in response to acute hypoxia and methacholine challenges, and responses will be compared to both sham and naive controls. Aim 3: Vagal ascending and descending nerve traffic will be recorded in fetuses instrumented with state-of-the-art cuff electrodes throughout fetal development and at birth. Custom time-series and spectral analysis software will be used to quantify changes in afferent/efferent vagal nerve signaling in response to pulmonary denervation, birth, and mechano- and chemosensory stimulations. The outcomes of these studies will fill in critical knowledge gaps of the role of interoception in lung development and function in a sheep model that has been a historically signature model of human perinatal lung development and physiology. Our proposed foundational work will inform future efforts focused on a variety of lung diseases in humans that originate during development, such as bronchopulmonary dysplasia, apnea of prematurity, sudden infant death syndrome, and asthma.