Dysregulation of breathing control through distinct molecular mechanisms of the brainstem cell population in neonatal sepsis

Abstract

Introduction Neonatal sepsis is triggered by a dysregulated immune response leading to systemic inflammation and life-threatening organ dysfunction. The systemic alterations and activation of immune cells will impact the activity of neural circuits, neural and non-neuronal cell populations, including those in the brainstem involved in breathing rhythm generation and pattern. Elucidating the extent to which inflammation modulates the CNS control of breathing has eluded us due to the lack of appropriate physiological maneuvers capable of interrogating specific brainstem circuits in the neonatal period. We perform novel respiratory testing in newborn pups with unrestrained plethysmography using distinct TLR ligands to test the hypothesis that distinct forms of systemic inflammation modulate brainstem circuits in different ways. Our goal was to investigate how different systemic inflammatory insults at the neonatal period affect the brainstem by determining the contribution of neurons, astrocytes, and microglia in the breathing plasticity response. Methods CD1 mice were I.P. treated at postnatal day 5 (PD5) with LPS, or Pam3CSK4(PAM), or saline (control). Using a whole-body plethysmography system, we assessed brainstem arousal reflex. Briefly, unrestrained pups were placed inside of the chamber individually, and submitted to a room air (RA) exposure for 06 min, followed by 01 min of a gas mixture of 97%N2/3%CO2 (anoxia) repeated 05 times. Ultrasonic vocalization recordings, a measure of brainstem-spinal circuit prolonged-forced expiration, were obtained with the Avisoft SASLab Pro software. Bulk brainstem and gene expression analysis of isolated neurons, astrocytes, and microglia were performed with Clariom™ S Assay mouse and Affymetrix GeneChip Mouse Gene ST 2.0 arrays. Differential gene expression was analyzed using a cutoff of 2-fold change and P-value of 0.01 and t-test in comparison to the control. Results Respiratory frequency during the baseline phase was 176 BPM in the control group, while was founded lower in the LPS and PAM group. Tidal volume (TVb) and minute ventilation (MVb) were also diminished in both groups compared to the control. The gasp latency, or time to recover after each anoxic episode, was prolonged impairing the autoresuscitation, especially, in the PAM treated group. Vocalization function was also affected by inflammation presence resulting in a reduced number of calls per min. A robust gene modulation in the brainstem bulk of PD5 pups was caused in both inflammatory insults. The enrichment analysis showed activation of T helper cells whereas CIBERSORT analysis indicated enrichment of Treg and B cells memory. The molecular pathways activated in neurons, astrocytes, and microglia were founded distinct and specific to the TLR ligand while astrocytes exhibited a lack of responsiveness caused by PAM. Conclusions We conclude that TRLs ligands activate specific neural mechanisms that will detrimentally affect ventilatory response and the ability to autoresuscitate via a central mechanism in the course of neonatal sepsis.