RNA sequencing to profile transcriptional changes within the medullary raphe: potential mechanisms of central neuroplasticity driving the recovery of eupneic ventilation after bilateral carotid body denervation (713.7)

Abstract

Bilateral carotid body denervation (CBD) in rats elicits eupneic (resting) hypoventilation 1‐2 d post‐CBD, which relatively rapidly returns to baseline values (14‐15 d). We have previously shown increases in brainstem serotonin (5‐HT) turnover and the 5‐HT transporter (SERT) acutely after CBD, leading to the hypothesis that the recovery of eupneic ventilation following CBD may be associated and/or initiated by activation of the medullary raphe nuclei (MRN). To test this hypothesis, we collected medullary raphe tissue punches (~1mm3) from rats euthanized acutely (1‐2 days) after sham (n=11) or CBD (n=10), or 15 days after sham (n=10) or CBD (n=10) surgery. Eupneic PaCO2 was increased acutely (p<0.001) and chronically (p<0.001) after CBD compared to pre‐CBD control, but PaCO2 15 days post‐CBD was not different than time‐control sham rats demonstrating recovery following CBD. Total RNA was collected using a phenol‐chloroform extraction protocol and quantified/qualified using an Agilent Bioanalyzer. 100 – 600 ng of total RNA was collected with an integrity number of 7 or greater for all samples, indicating high RNA quality. 12 cDNA libraries were created (3 or 4 per group x 4 groups) using the Illumina TruSeq RNA Sample Prep LS protocol for mRNA sequencing, from which all 12 libraries yielded pure cDNA. Ongoing mRNA sequencing will allow for the eventual coupling of our comprehensive physiologic measurements with raphe gene expression to provide unique insights into potential mechanisms of recovery or plasticity ventilatory control after CBD.Grant Funding Source: Supported by NIHHL097033, T32HL007852.