A trilogy of stressors in the neonatal intensive care unit: towards therapy for preterm adversity

Thumbnail Image
Dias Casacao, Maria Luis
Journal Title
Journal ISSN
Volume Title
University College Cork
Published Version
Research Projects
Organizational Units
Journal Issue
Premature infants are born with immature breathing network and an innate immune system that responds differently to that of infants born full-term. All infants born less than 28 weeks of gestation develop apnoea of prematurity-related symptoms, which decreases to about 20% of infants born at 34 weeks of gestation and to less than 10% of those infants born beyond 34 weeks of gestation. Premature infants are not only at risk of developing breathing disorders but also at increased risk of infection due to early life vulnerability (the earlier a baby is born, the more likely is to have health problems due to immature organ development). Gram-positive (GP) infections are the most common type of late onset infection in preterm infants. Activation of specific toll-like receptors (TLRs) is reported to modulate cardiorespiratory responses during infection and may play a key role in driving homeostatic instability observed during sepsis. Firstly, we sought to characterise the expression of TLRs in the brainstem, adrenal gland and in the diaphragm respiratory muscle in naïve rats during our developmental period of interest (postnatal day (PND) 3 and PND13). These studies demonstrated mRNA expression of these receptors at PND3. TLR expression fluctuated in early life depending on the subtype and tissue examined with a relative decrease in some of the mRNA expression at PND13; TLR1, 2, 4, 6, 9 and NOD2 in sternohyoid muscle, TLR1, 2, 4 and 6 in diaphragm muscle, TLR2 in adrenal glands and NOD2 in brainstem and spinal cord, but a relative increase in mRNA expression of CX3CR1 in brain and brainstem, TLR2 and 9, IL1R1, in brain and TLR2 and TLR9 in spinal cord. Sex differences were revealed in mRNA expression of TLR9 in brain and NOD2 and IL1R1 in brainstem with upregulation of expression in males. These results relating TLRs and postnatal development suggest a developmental regulation of the immune system. Given these results, we reasoned that oxygen dysregulation coupled with GP bacterial immune stimulation would modulate redox sensitive genes and TLR expression that could alter hormonal expression and impinge on respiratory function in a sex-specific manner. To test this, we developed a novel neonatal rat model in which male and female neonatal rats were exposed to intermittent hypoxia, normoxia and hyperoxia from PND3 for 10 days, followed by combined administration of GP bacterial proteins lipoteichoic acid (LTA) and peptidoglycan (PGN). This model sought to mimic physiological challenges encountered by infants born preterm. Hypoxia challenges during the intermittent hypoxia and hyperoxia (IHH) protocol, induced a significant peripheral oxygen desaturation in treated animals. LTA and PGN (3mg and 5mg, respectively) evoked a significant immune response in PND13 rat pups when measured 3 hours post administration. Serum cytokine analysis revealed LTA&PGN triggered an increase in CCL2, IL-1α, IL-1ß, IL-5, G-CSF, IL-13, CCL3, Gro/KC, CXCL10, CX3CL1, CXCL2, IL-10, IFNy, leptin, VEGF, IL-17A and TNF-α serum concentration compared to vehicle. Interestingly, IL-1ß, Gro/KC, IL-10 and leptin expression were upregulated with combined IHH and LTA&PGN exposure. Respiratory function demonstrated an overall decrease in breathing frequency that was mainly impacted by LTA&PGN administration due to an increase in expiratory time. A decrease in minute ventilation was reported with LTA&PGN however, regarding the metabolic function, the ventilatory equivalent for carbon dioxide was similar across the groups, which is consistent with normal pH levels obtained. Additionally, a mild response to the GP challenge in the late periods of hypoxia were associated with decreased number of gasps with IHH. We analysed mRNA expression of TLRs and redox modulated genes using real-time polymerase chain reaction (RT-PCR) in brainstem, diaphragm muscle and adrenal glands. Brainstem gene expression was similar across groups. In adrenal glands tissue, there was an overall upregulation of TLRs mRNA expression with IHH exposure, except for TLR6. Moreover, TLR2 mRNA expression was upregulated with IHH compared to Sham groups, in males compared to females and in LTA&PGN compared to vehicle. An inverse trend from that of adrenal glands was reported in diaphragm muscle. We also analysed redox modulated proteins in serum using bioassays to detect 8-OHdG, 8-iso-PGF2α, AOPP and SOD in plasma. No differences were observed with IHH and LTA&PGN. However, sex differences were found in 8-iso-PGF2α and AOPP redox proteins, with upregulated expression in males compared to females. Finally, we sought to characterise the postnatal development in our animal model using a battery of developmental and motor assessments. These studies demonstrated a delay in pinnae detachment in IHH pups, a decrease in time to righting with IHH and an increase in motor and locomotion faults in IHH females. Tactile stimulation was decrease with IHH suggesting a delay in brainstem reflexes. IHH treated males presented with an increased expression of stress and anxiety-related behaviours illustrated by increased time spent in the corners of the open-field test with LTA&PGN and distance travelled with IHH, and decreased time spent in open arms in the elevated plus maze experiment in males compared to females. Altogether, these results suggest that early life stress can profoundly impact the expression of TLRs and redox genes in adrenal glands, impact the expression of cytokines such as leptin and alter development, motor coordination, stress levels in this novel neonatal rat model of early life stress. In conclusion, these studies specifically target the gaps in knowledge of the pathophysiological alterations experienced in prematurely born babies who present with impaired breathing function and contract GP infection as mimicked in this novel animal model. The results presented disclose novel insights on the physiology and hormonal alterations that these babies could face in similar conditions, with potential to positively contribute to the field of study by enlightening future targets of research. Future directions relying on the spatial characterisation of TLRs and leptin receptors and possible long-term influences on behavioural performance would also be helpful to better characterise the model.
Early life , Breathing function , Infection
Dias Casacao, M. L. 2023. A trilogy of stressors in the neonatal intensive care unit: towards therapy for preterm adversity. PhD Thesis, University College Cork.
Link to publisher’s version