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- 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.
- ItemOptimisation of gene editing for cystic fibrosis(University College Cork, 2020-06-06) Mention, Karen; Harrison, Patrick; Scallan, Martina; Cystic Fibrosis TrustCystic Fibrosis (CF) is a recessive genetic disease caused by mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. To date, 352 variants in the CFTR gene have been shown to be CF-causing. CF is the most common genetic disease in Caucasian population, with an estimation of about 70,000 to 100,000 people living with CF worldwide. The disease results in premature death at a median age of 44 years old, with patients dying mostly from end-stage lung disease as a consequence of chronic lung infections. There is no cure for CF, but there are a range of drugs to treat CF symptoms. Over the last nine years, some small molecule drugs called modulators, were designed to improve the processing and function of the CFTR protein slowing the progression of the disease for more than 90% of CF-patients. Even though those modulators revolutionised CF treatment, the cost for those treatments are expensive, cumbersome and there are still 10% of patients with no specific drug. Indeed, some CF-causing mutations, classified as Class I variants, result in expression of little or no CFTR protein; protein modulator therapies are ineffective for patients suffering from such mutations. The variant W1282X is one of them. The W1282X variant is the 6th most common CF-causing variant, concerning 2.5% of CF patients, moreover, it is the 2nd most common class I variant. Since the discovery of the CFTR gene in 1989, it was expected that being able to treat the genetic problem, could lead to a treatment for CF. Since then, multiple clinical trials for CFTR cDNA addition have been performed, unsuccessfully. However, since the discovery of programmable nucleases, for gene editing, new hopes for CF gene therapy emerged. Indeed, some clinical trials are in process for other diseases such as Leber’s congenital amaurosis, haemophilia B or mucopolysaccharidosis I and II. The goal of this project was to compare four different techniques to correct the W1282X mutation, either by itself using homology-directed repair (HDR) and base editing, or as a superexon to correct this mutation and all the ones downstream. The purpose was to determine if there was one technique that was optimal for CF correction. Targeting single mutations, the results showed that high correction efficiencies (around 20% with SpCas9 HDR and base editing and 8% with AsCas12a HDR) could be achieved, and the corrections led to accumulation of corrected mRNA (50% for AsCas12a HDR and Base editing to 60% for SpCas9 HDR). In addition, CFTR protein expression could also be observed in AsCas12a-edited samples. However, using HDR, a large amount of indels could be detected, disrupting the CFTR gene in non-corrected alleles. Moreover, base editing showed formation of by-stander modifications within the window of editing. Using a superexon for CFTR correction, the homology-independent targeted integration (HITI) technique showed an intermediate level of correction efficiency of about 6% in 16HBE14o- cells after selection, leading to about 8% of corrected mRNA. Using HDR to replace a large DNA sequence, the efficiency without selection appeared to be low with about 0.02% of mRNA correction; editing at DNA level could not be determined for this technique in the cell lines available. Even though the efficiencies appeared to be lower using a superexon, the systems seemed to be safer with indels localised in introns. Using those data, it could be possible to have a clear understanding of different gene editing techniques to correct the W1282X mutation. Those techniques could be used for other mutations as well as for other genetic diseases. With further optimisation, one or many of these techniques could be tested on CF animal models to provide safety data for a potential future use in the clinic for CF-patients.