ItemA trilogy of stressors in the neonatal intensive care unit: towards therapy for preterm adversity(University College Cork, 2023) Dias Casacao, Maria Luis; McDonald, Fiona; Carolyn Sifton Foundation; Science Foundation IrelandPremature 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. ItemAn exploration into the effects of high altitude exposure on the neurovascular coupling response(University College Cork, 2022) Leacy, Jack K.; O'Halloran, Ken; Day, Trevor A.; University College Cork; Mount Royal University; Natural Sciences and Engineering Research Council of CanadaIntroduction The neural infrastructure within cerebral tissue is both elaborate and metabolically volatile. Owing to limitations in energy substrate storage capacity, the neural nexus relies upon continuous perfusion for the delivery of essential nutrients. Vital substrates which are transported within the blood help to support energy-dependent metabolic processes throughout the central nervous system at a global and regional level. Despite constituting ≈2% of total body mass, the brain receives a disproportionate ≈20% of total cardiac output. The physiological phenomenon which describes the intimate link between local neuronal activity and regional cerebral blood flow is termed neurovascular coupling (NVC). This phenomenon has been observed across all cerebral structures wherein dynamic changes in local metabolic activity are met with a concomitant increase in local perfusion of the active tissue. A vast body of work has assessed the translational implications of NVC for neurophysiological function and behavioural outcomes across several pathologies (aging, neurodegenerative disease, autoimmune disorders, trauma and metabolic disorders). Thesis Aims Herein, we sought to characterise the effects of high altitude (HA) exposure on NVC in healthy human volunteers encompassing different ascent paradigms (chapters 3 and 4). HA exposure is a pernicious multimodal stressor among habitual lowlanders, which necessitates an integrative physiological acclimatisation to sustain homeostasis and overall health and function. This environmental stressor has pronounced effects on other components of cerebral blood flow control. Prior to this thesis there was a dearth of literature regarding the effects of HA exposure on NVC. In addition, we conducted a complementary laboratory-based investigation which examined the degree of variance within the NVC response attributable to either age and/or sex (chapter 2). This comprehensive study was essential with respect to appreciating the interaction between participant demographics (age, sex) and NVC. Demographic heterogeneity is an inherent component of many HA research expedition. We therefore determined that it was of paramount importance to ascertain the magnitude to which heterogeneity influences our primary physiological measures. Methods Transcranial Doppler ultrasound (TCD) was employed to provide non-invasive measures of cerebral blood velocity. NVC was indexed as the change in posterior cerebral artery velocity (PCAv) during intermittent visual stimulation (30s on/off; 6Hz) in all experiments. This technique is widely used within the literature, as visual stimulation mediates an increase in metabolic activity of the visual cortex, a neural territory perfused by the PCA. Healthy human volunteers were recruited for studies described in chapter 2 (n=125, 41 male), 3 (n=10, 4 male) and 4 (n=12, 7 male). Arterial blood draws were sampled from the radial artery during HA expeditions (chapter 3 and 4), providing and index of ventilatory and acid-base acclimatisation status within each participant. Ascent profiles differed between chapters 3 and 4 relevant to the experimental question. Key results and conclusions Collectively, our results demonstrate that approximately 0-6.1% of the variance across several NVC metrics are attributable to either age and/or sex (chapter 2). Moreover, the magnitude of the haemodynamic response across several NVC metrics was remarkably intact following incremental and acute ascent to 4240m and 3800m respectively (chapters 3 and 4 respectively). Taken in conjunction, this thesis reveals that: (1) Neither age nor sex greatly affect the magnitude of the NVC response in healthy human volunteers (21-66yrs old) and (2) NVC response magnitude remains remarkably intact across multiple HA exposure paradigms in habitual lowlanders. ItemInvestigating the role of interleukin-6 in neuronal dysfunction in the mdx mouse model of Duchenne muscular dystrophy(University College Cork, 2022-01-07) Stephenson, Kimberley Alix; O'Malley, Dervla; Rae, Mark; Health Research BoardDuchenne Muscular Dystrophy (DMD) is a fatal musculoskeletal disorder that results in a loss of dystrophin in muscle fibres, leading to progressive immobility, chronic inflammation and premature death. Inflammation is characterised by increased circulating levels of pro-inflammatory cytokines such as IL-6. However, it is apparent that dystrophin not only functions in skeletal muscle, but in the CNS as well, with an increased incidence of cognitive impairment and deficits in verbal, short-term and working memory in individuals with DMD. The hippocampus is critical for learning and the formation and consolidation of memories and can be modified by neuroimmune cytokines, such as IL-6. The aim of this programme of research was to investigate the possible contribution of the neuroimmune molecule, IL-6 in cognitive impairment associated with loss of the structural protein, dystrophin. Initially, we used the dystrophic mdx mouse to investigate what impact loss of dystrophin had on normal hippocampal function. Significantly, LTP, the molecular correlate of learning and memory was shown to be decreased in mdx mice, which has been linked to memory dysfunction. Moreover, changes in the density and structure of mdx hippocampal neurons and glia was observed over time. Subsequently, we examined the possible effects of IL-6 on hippocampal function in mdx mice. IL-6 and IL-6R expression were altered in specific regions in the hippocampus, which underpins function relating to learning and memory. Indeed, in vivo blockade of IL-6-mediated signalling in the CNS of mdx mice restored LTP, although at this age, it did not impact on learning behaviours. These studies have elucidated previously unknown cellular and molecular changes in the hippocampus of dystrophin-deficient mice. Moreover, we have linked the neuromodulatory cytokine, IL-6, with the observed pathophysiology. ItemThe role of renal afferent signalling in chronic intermittent hypoxia-induced sympathoexcitation and hypertension(University College Cork, 2021) AlMarabeh, Sara; O'Halloran, Ken D.; Abdulla, Mohammed; University of Jordan; University College CorkIntroduction: Sensory inputs from the kidney induce sympatho-excitation, and are integrated in brainstem regions receiving protective sensory inputs from high- and low-pressure baroreceptors. Blunted baroreflex control of renal sympathetic nerve activity (RSNA) was revealed in hypertension models that involve renal inflammation. Suppression of inflammation restored the normal baroreflex control of RSNA in some of these models, suggesting that renal inflammation impairs baroreflex control of blood pressure through the disruption of renal afferent nerve signalling. Renal oxidative stress and inflammation are evident following exposure to chronic intermittent hypoxia (CIH) in addition to blunted baroreflex control of heart rate. However, little information is available about the baroreflex control of RSNA. In addition, because kidney injury disrupts renal afferent nerve signalling, changes in the renorenal reflex control of sympathetic outflow may occur following exposure to CIH. Therefore, understanding the stage at which baroreflexes and the reno-renal reflex are altered is required to explore the mechanisms that contribute to the early CIH-induced sympathetic hyperactivity and the onset of hypertension. Methods: Following exposure to CIH or normoxia, baroreflexes were examined under anaesthesia. Kidney excretory function was measured during the assessment of low-pressure baroreflex by volume expansion (VE). Baroreflexes were assessed before and after blockade of renal TRPV1 channels. Moreover, to investigate if the excitatory reno-renal reflex contributes to sympathetic over-activity in CIH, renal afferent nerves located in the renal pelvic wall were chemically stimulated by bradykinin and capsaicin, or inhibited by bradykinin receptor type 1 (BK1R) and/or 2 (BK2R) blockers, and cardiovascular and RSNA responses were measured. Renal histology, inflammation and oxidative stress biomarkers were assessed. Results: CIH-exposed rats were hypertensive with elevated RSNA, with no evidence of glomerular hypertrophy or renal inflammation and oxidative stress. Water and sodium excretion were increased following CIH exposure. However, diuresis and natriuresis during VE were attenuated in CIH-exposed rats despite preservation of the progressive decrease in RSNA during VE, suggesting that altered kidney excretory function in CIH was independent of neural control. The increase in atrial natriuretic peptide during VE was attenuated in CIH. Assessment of the high-pressure baroreflex revealed decreased slope in CIH-exposed rats with substantial hypertension, but not when hypertension was modest. Diuresis and natriuresis during VE were enhanced in CIH-exposed and sham rats following the intra-renal blockade of TPRV1 channels, suggesting a role for renal TRPV1 in the control of renal excretory function. However, TRPV1 protein expression in the kidney was unchanged and TRPV1 activation by intra-renal pelvic infusion of capsaicin induced a similar sympatho-excitation in sham and CIH-exposed rats. Moreover, sympatho-excitation during intra-renal pelvic infusion of bradykinin was suppressed in CIH-exposed rats. This was associated with 53% decreased expression of BK2R in the renal pelvic wall of CIH-exposed rats compared with sham rats. Inhibition of renal bradykinin receptors did not affect cardiovascular parameters or RSNA in sham and CIH-exposed rats. Conclusion: Our findings show no evidence of an excitatory reno-renal reflex driving sympathetic hyperactivity and the onset of hypertension in CIH. This was revealed by the absence of renal pathology despite the presence of a hypertensive phenotype. Moreover, the findings indicate suppressed rather than exacerbated sympatho-excitation in CIH-exposed rats in response to bradykinin. In addition, the baroreflex control of RSNA was maintained in CIHexposed rats with modest hypertension, indicating that blunted baroreflex control is not obligatory for the onset of hypertension in CIH. Overall, renal injury appears to develop after the progressive elevation of blood pressure, although it may also develop in circumstances of exposure to severe CIH, suggesting that chronic kidney disease, frequently observed concomitant with obstructive sleep apnoea (OSA), may be mitigated if OSA is controlled at an early stage. ItemThe role of NADPH oxidase-2 in chronic intermittent hypoxia-induced respiratory system dysfunction(University College Cork, 2020-12) Drummond, Sarah E.; O'Halloran, Ken D.; Healy, VincentObstructive sleep apnoea syndrome (OSAS) is characterised by exposure to chronic intermittent hypoxia (CIH) as a consequence of repetitive occlusions of the upper airway in patients during sleep. Exposure to CIH evokes redox changes which culminate in impaired upper airway and diaphragm muscle function. Excessive reactive oxygen species (ROS) are also associated with aberrant respiratory plasticity, which manifests as destabilised breathing during sleep. There is a paucity of information regarding the molecular mechanisms underlying these effects. We sought to investigate the putative role of the superoxide-generating NADPH oxidase-2 (NOX2) enzyme in CIH-induced respiratory muscle dysfunction and respiratory mal-adaptation. A mouse model of CIH was generated by the cycling of gas from normoxia (21% O2) for 210 seconds to hypoxia (5% O2 at the nadir) over 90 seconds for 8hr/day for 2 weeks. Adult male (C57BL/6J) mice were assigned to one of 5 groups: normoxic controls, CIH-exposed, CIH + apocynin (NOX2 inhibitor, 2mM) given in the drinking water throughout the exposure to CIH, and NOX2 null (B6.129S-Cybbtm1Din/J) assigned to a sham or CIH exposure. On day 15, whole body plethysmography (WBP) was used to measure breathing parameters on a breath-by-breath basis in room air and in response to chemostimulation. An apnoea was defined as ≥ 2 missed breaths. Sternohyoid and diaphragm muscle contractile function was examined ex vivo. Gene expression was examined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Western blot was used to measure protein expression. NOX enzyme activity and indices of oxidative stress were determined using spectrophotometric assays. Basal minute ventilation (V̇I) was unchanged following 2 weeks of exposure to CIH, however the number of apnoeas per hour was significantly increased compared with sham-exposed mice. Apocynin intervention significantly reduced the frequency of apnoeas compared with the CIH-exposed group. Apnoea index was increased in NOX2 null mice exposed to CIH, reminiscent of that observed in wild‐type mice. Exposure to CIH resulted in severe sternohyoid and diaphragm muscle weakness, evidenced by a ~45% reduction in the force-generating capacity of these respiratory muscles compared with sham. Exposure to CIH increased NOX enzyme activity in the sternohyoid, with no alteration to the gene or protein expression of NOX subunits. In contrast, exposure to CIH increased the protein and mRNA expression of NOX4 in the diaphragm, while NOX activity and expression of all other NOX subunits remained unchanged. No indication of overt oxidative stress was observed in the diaphragm or sternohyoid muscle following exposure to CIH. Apocynin treatment and NOX2 gene knock-out completely prevented CIH‐induced diaphragm and sternohyoid muscle weakness. Exposure to CIH increased the mRNA expression of genes relating to autophagy, atrophy and muscle differentiation in the diaphragm in a NOX2-dependent manner; these CIH-induced responses were not observed in the sternohyoid. Mice show signs of profound respiratory muscle dysfunction following exposure to a mild-to-moderate paradigm of CIH. The putative NOX inhibitor, apocynin, prevents CIH-induced respiratory muscle weakness. Moreover, studies in NOX2 null mice reveal that NOX2 is obligatory for CIH-induced respiratory muscle weakness. The mechanisms underpinning CIH-induced muscle weakness likely differ between sternohyoid and diaphragm muscle, evidenced by varied molecular responses to CIH, which may relate to differences in fibre-type expression. CIH-induced respiratory muscle weakness may contribute to upper airway obstruction and impaired swallow and cough, relevant to OSAS. The reduction in apnoea frequency following treatment with apocynin, but not NOX2 knock-out, implicates ROS (that are not NOX2-derived) in the manifestation of CIH-induced respiratory disturbances. CIH-induced increase in the propensity for apnoea may be of clinical relevance as it may underpin progression in the severity of OSAS pathology (i.e. mild-to-moderate-to-severe OSAS). Our results have implications for human OSAS and point to antioxidant intervention, potentially targeting NOX2 blockade, as a potential therapeutic strategy.