Florey Department of Neuroscience and Mental Health - Theses

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    The influence of cocaine on cortical neurons
    Murphy, Sean Christopher ( 2023-11)
    Cocaine is a powerfully addictive stimulant drug which produces short-term euphoria in users by highjacking the brain’s reward system. By binding to synaptic transporters, cocaine prevents the removal of specific neuromodulators, such as dopamine, serotonin and noradrenaline from the synaptic cleft. This leads to elevated extracellular levels of these neuromodulators which leads to re-activation of the synapse and neuronal hyper-excitability. Despite the prevalence and dramatic effects of cocaine use, the functional effects of cocaine exposure on the activity of neurons in the intact brain are poorly understood. To fill this gap in knowledge, my Ph.D research investigated the influence of cocaine on neural activity, presented in this Thesis as two projects. First, I investigated how cocaine influences the processing of information within the sensory cortex. Using voltage recordings and two-photon dendritic and somatic Ca2+ imaging in vivo, I measured the influence of acute exposure of cocaine on layer 2/3 (L2/3) pyramidal neurons within the primary somatosensory cortex (S1). Here, cocaine dampened membrane potential state transitions and decreased spontaneous somatic action potentials. In contrast to the uniform decrease in background spontaneous activity, cocaine had a heterogenous influence on sensory encoding, increasing tactile-evoked responses in dendrites which did not typically encode sensory information, and decreasing those dendrites which were more reliable sensory-encoders. Combined, these findings illustrate that exposure to cocaine has a heterogenous influence on cortical neurons, leading to the dampening of background spontaneous activity and selective filtering of sensory input. Second, I investigated how cocaine influences the activity of thalamic axonal projections, and the influence this has on cortical neurons in a different context. The thalamus acts as a central hub and is perfectly positioned to drive the modulation of cortical activity following cocaine exposure. Two photon calcium imaging was performed in both the higher-order (POm) thalamic axonal projections and layer 2/3 (L2/3) pyramidal neurons within the primary somatosensory cortex (S1) of awake mice during cocaine administration. Single exposure to cocaine caused a significant increase in the thalamic and cortical signaling in S1, and chemogenetic dampening of POm suggested the thalamus may predominantly drive the increase in cortical activity during acute cocaine. Combined, the results of these studies will provide invaluable insight into the effects of cocaine on brain functioning and will help us unravel the neural correlates of drug addiction.
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    Regulation of Ferroptosis Signalling Pathways: Implications for Alzheimer's Disease
    JAKARIA, MD ( 2023-12)
    Background and aims: The natural history of Alzheimer's disease (AD) involves the formation of extracellular amyloid plaques followed by intracellular neurofibrillary tangles that are associated with neurodegeneration, leading to cognitive deterioration and death. The mechanism of neurodegeneration in AD is not fully understood. Ferroptosis is a form of regulated cell death due to uncontrolled phospholipid peroxidation, which has been implicated as a mechanism of neurodegeneration in AD. Amyloid precursor protein (APP) has been considered the central protein involved in the pathogenesis of AD. Amyloidogenic processing of APP via beta-secretase (BACE-1) followed by gamma-secretase generates amyloid-beta peptides, the major component of amyloid-beta plaques. Alternatively, non-amyloidogenic APP processing via alpha-secretase generates soluble amyloid precursor protein alpha (sAPPalpha) that protects against rotenone-induced toxicological insults, serum deprivation and hypoxia. The role of APP and its processing and APP-associated signalling in ferroptosis has yet to be investigated. The first part of my thesis project examined the impact of APP and its processing on ferroptosis. Growing evidence suggests that vitamin A (retinol) and its metabolite all-trans retinoic acid are modulators of APP processing and neuroprotectors in pre-clinical and clinical AD models. The second aim of the project was to examine whether retinol and its metabolites affect ferroptosis sensitivity. Brain-derived neurotrophic factor-tyrosine kinase B receptor (BDNF/TrkB) signalling has also been shown to modulate APP processing and protect against neurodegeneration in pre-clinical and clinical AD models. The final aim of the project was to characterise the role of BDNF/TrkB signalling on ferroptosis. Methods: We used a cell culture model of ferroptosis to investigate these thesis projects. Several inducers were used to cause ferroptosis in diverse neuronal and non-neuronal cell lines. This thesis used various molecular biology, biochemical and cell-free techniques to investigate ferroptosis regulation. Results: The first aim of the thesis found that neuronal and non-neuronal cell lines expressing APP desensitise ferroptosis. APP expression enhanced the heme-Nrf2-GPX4 axis, the central cellular antioxidant defence, which explains APP desensitisation of ferroptosis. By investigating the role of APP processing on ferroptosis, we found that inhibiting alpha-secretase sensitises cells to ferroptosis, possibly via downregulating GPX4. However, we did not observe the involvement of APP-processed products in the pro-ferroptotic effect of alpha-secretase inhibition. By investigating the second aim, we showed retinol and its major metabolites, all-trans-retinal and all-trans-retinoic acid, confer superior anti-ferroptotic activity than alpha-tocopherol (an endogenous anti-ferroptotic vitamin), most likely acting as radical trapping antioxidant (RTA) agents, not by interfering with APP processing. Conversely, anhydroretinol (an antagonist of retinol uptake and functions) sensitised ferroptosis, suggesting a role for retinol signalling against ferroptosis at physiological levels. The final part of the thesis showed the role of several TrkB modulators on ferroptotic susceptibility. The modulators of the TrkB receptor, including an agonist, antagonist and inhibitor, were found not to affect RSL3-induced ferroptosis via modulating the TrkB receptor signalling, suggesting that TrkB modulation might not directly involve ferroptosis in our model. In contrast, several modulators of the TrkB receptor conferred anti-ferroptotic activity, which may be explained by their inherent RTA properties. Conclusion: The thesis identified several novel regulatory pathways involving ferroptosis and neurodegeneration that could be targeted for AD therapy.
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    Investigating the circuit mechanisms of the Solitary Tract Nucleus
    Thek, Kimberly Rose ( 2023-10)
    Maintaining physiological homeostasis requires the brain to accurately receive and process information on the state of the body. Interoception, the sense of the internal state of the body, is a fundamental component of homeostasis that links the body’s physiological needs with both conscious, and unconscious, feelings and behaviour. Understanding how the brain makes sense of the internal state of the body is a major challenge due to the enormous diversity of sensory modalities. To determine the neural basis of interoception, we must first define the features of the circuit mechanisms by which sensory signals are processed and integrated in the maintenance of homeostasis. The solitary tract nucleus (NTS) is the initial site for central processing of sensory information relating to the internal state of the body. A highly heterogeneous nucleus, the cellular and circuit mechanisms of sensory processing at this initial site are not well defined. In this thesis, I aimed to first classify the electrophysiological features – groups of distinct electrophysiological characteristics, that define groups of NTS neurons. I then went on to investigate plasticity of these features via a perturbation of the vagus nerve, the major source of afferent sensory input to the NTS. I developed a semi-supervised machine learning method to define groups of neurons with electrophysiological characteristics that did not correlate with homogeneous neurochemistry. Following chronic vagus nerve stimulation (VNS) in Sprague-Dawley rats, active firing patterns of NTS neurons were shifted from irregular burst firing, towards tonic firing, identified via a multimodal analysis of electrophysiological characteristics. Plasticity was also identified in NTS neurons following chronic VNS in a rodent model of genetic absence epilepsy, however the effect was not sufficient to reduce hyperexcitability of neurons to a non-pathological control. Throughout this thesis, I present a case for investigating neural mechanisms through the lens of homeostasis, whereby multimodal features, not individual characteristics, define classification of neurons and their function within circuitry.
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    Mimicking the effects of gene x environment interaction with small non-coding RNAs associated with anxiety and depression
    Kuznetsova, Maria ( 2023-06)
    Depression and anxiety are debilitating psychiatric disorders, which significantly depend on genetic predisposition and changing environments. One in three patients is resistant to current antidepressant treatment, highlighting the need for novel therapeutics to improve patient outcomes. Understanding how combination of genetic and environmental factors contribute to the development of depression and anxiety through microRNAs will aid the development of novel therapeutics for affective disorders. During my PhD, I explored how combinations of genetic and environmental factors affect expression of brain miRNAs in a mouse model of treatment-resistant depression (TRD). In the first part of my PhD project, I described miRNA composition in the hippocampus of the serotonin transporter knock-out (5-HTT KO) mice compared to wild-type mice in standard housing conditions. 5-HTT KO mice have increased depression-like behaviour and do not respond to classic antidepressant treatment, which make them a good model of TRD. This is the first study describing miRNA profiles in the brain tissues of a mouse model of TRD. We discovered novel miRNAs, which could be used as markers of TRD. The enrichment analysis of target genes of these miRNAs revealed pathways relevant to stress response, highlighting the importance of changing environments in the development of TRD. Next, I described miRNA profiles in 5-HTT KO mice after stress and exercise, which was the first study on how gene x environment interactions affect miRNA composition in the brain of mice genetically predisposed to TRD. Based on these data, I targeted several miRNAs using novel approaches of miRNA modulation to mimic beneficial effects of exercise and stress-reduction to develop new treatment for TRD. I observed a significant antidepressant-like effect after treatment with miRNA mimic, which proved that miRNAs can not only be a marker of depression but are themselves targets for the treatment of depression. The follow-up bioinformatic analysis revealed that miRNA mimic treatment enhanced neuroplasticity and neurotrophic support by activating cascade of genes, which protect or rescue neurons that are vulnerable after stress or other insults. This study provided the first description of miRNA alteration in a mouse model with serotonergic dysfunction and highlighted the potential of these miRNAs as therapeutic targets for TRD. These results provide further evidence to highlight the importance of considering gene-environment interactions in the management and treatment of affective disorders. Ultimately, these findings will aid in the development of genetically-informed precision medicine for serotonergically-dependent refractory mental illness.
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    Interrogating TAM receptor activation for therapeutic benefit in multiple sclerosis
    Asadian, Negar ( 2023-05)
    Multiple sclerosis is an autoimmune neurodegenerative disease characterized by demyelination and axonal damage in the central nervous system (CNS). Remyelination plays a crucial role in axonal protection and functional recovery. The GAS6 protein has emerged as a promising candidate for enhancing remyelination. This thesis aims to uncover the underlying mechanisms through which GAS6 exerts its pro-myelinating effect. Additionally, it delves into the pharmacokinetic properties of this protein, emphasizing the significance of gamma-carboxylation in the GAS6 GLA domain in mediating myelination in vitro, along with its residence time in the CNS. The latter part of this doctoral work demonstrates the utilization of the PEGylation approach to extend the brief CNS residence time of the GAS6 protein. The data from this project suggest that the Tyro3 receptor significantly contributes to GAS6's pro-myelinating effect in the CNS after demyelination. This observation appears to be partly driven by mature oligodendrocytes and seems largely independent of the inflammatory response. This research also underscores the importance of gamma-carboxylation within the GAS6 GLA domain for its pro-myelinating effect in vitro. Additionally, it reveals that GAS6 exhibits a short residence time of less than two hours in the murine brain. Using the PEGylation approach, a bioactive variant of this protein with an extended CNS residence time was successfully generated. In summary, this work identifies the Tyro3 receptor as a potential target for the GAS6 pro-myelinating effect and sheds light on some pharmacokinetic properties of the GAS6 protein, particularly highlighting the significance of its post-translational modifications and brief CNS residence time.
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    Sperm noncoding RNAs as mediators of paternal epigenetic inheritance modulating offspring affective and social behaviours
    Barbosa Hoffmann, Lucas ( 2023-06)
    Background: Studies have shown that paternal environmental conditions prior to conception can influence the innate behaviours of their offspring, and the evolutionary impacts of such intergenerational effects are therefore of considerable interest. Epigenetic mechanisms have been shown to underlie this inheritance, as the microinjection of sperm small noncoding RNAs into fertilised oocytes induces reprogramming of the early embryo, which is thought to be responsible for the differences observed in adult phenotype. Our group previously showed in a mouse model of daily stress that glucocorticoid treatment of adult male breeders prior to conception leads to increased anxiety-related behaviours in male offspring, and this accompanies changes in the paternal sperm small noncoding RNA profile. Additionally, in a model of paternal running wheel voluntary exercise preconception, our group observed lower anxiety levels and a more robust fear extinction memory in the male offspring, as well as changes in paternal sperm small noncoding RNA expression. Aims: In this study, we aimed to understand the transgenerational effects of paternal stress exposure on the social behaviour of the male progeny and its potential influence on reproductive success by analysing its effects on social reward, male attractiveness and social dominance. We also assessed the paternal sperm long noncoding RNA profile following glucocorticoid treatment or running wheel voluntary exercise. We used CaptureSeq, a sequencing technique that is more sensitive than the ones used in other studies in the field. We next sought to determine the role of sperm long noncoding RNAs by microinjecting them into fertilised oocytes. Results: We report that paternal corticosterone-treatment was associated with increased display of subordination towards other male mice. Those mice were unexpectedly more attractive to female mice while expressing reduced levels of the key rodent pheromone Darcin, contrary to its conventional role in driving female attraction. Furthermore, no overt differences of the prefrontal cortex transcriptome were found in the offspring, implying that peripheral mechanisms are likely contributing to the phenotypic differences. No transgenerational differences were observed. Paternal corticosterone exposure led to dysregulation of sperm long noncoding RNA expression, which encompassed lncRNAs, circular RNAs and transposable elements. Although they have poor functional annotation, bioinformatic approaches indicated their expression in the brain, as well as their potential in regulating brain function. Running wheel exercise led to hundreds of downregulated lncRNAs, as well as transposable elements, and bioinformatic strategies predicted their function in biological processes, such as cell adhesion. Lastly, we separated and isolated the sperm long noncoding RNA population after glucocorticoid exposure and performed microinjections into fertilised oocytes. We observed that the resulting adult offspring had lower body weight and altered behavioural responses in the light-dark box and Porsolt swim test. Conclusion: Our findings highlight the potential of paternal stress to affect intergenerational (mal)adaptive responses. They also provide insights into the potential biology of long noncoding RNAs and highlight that efforts to annotate their function are highly necessary for the understanding of the mechanisms underlying the epigenetic inheritance. We are also the first to show that voluntary exercise modulates sperm long noncoding RNAs expression.
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    Gastrointestinal complications of Parkinson's disease
    Chai, Xin-Yi ( 2023-07)
    Parkinson’s disease (PD) is a progressive neurodegenerative disorder that can also affect gastrointestinal (GI) function. External factors, such as chronic stress, have been shown to play a role in the development of PD. GI disorders, include constipation that is a common non-motor symptom of PD that affects around 80% of patients. Defecation is under voluntary control, beginning from centres in the brain, sending signals down the spinal cord to the lumbosacral defecation centre. However, the mechanisms involved in PD-associated constipation remain unknown. The toxin affecting catecholamine neurons, 6-hydroxydopamine (6-OHDA), is commonly used to model PD and causes constipation when injected into the medial forebrain bundle (MFB). In Chapter 3, I have shown that 6-OHDA rats developed motor dysfunction as well as deficits in colon motility, exhibited by a significantly increased bead expulsion time compared with shams. Furthermore, the number of contractions and propulsion of contents by 6-OHDA rats was significantly reduced in comparison to shams after the administration of capromorelin, a colokinetic that acts on the lumbosacral spinal cord, indicating that 6-OHDA animals have reduced responsiveness of the defecation circuits. Enteric neuropathy was observed in the distal colon, revealing that 6-OHDA lesioning of the MFB has downstream effects at the cellular level. Based on the results of Chapter 3, it leaves open two questions that are addressed in Chapter 4: is the responsiveness of neurons downstream from the spinal defecation centres affected by 6-OHDA and could leakage of 6-OHDA into the periphery contribute to the changed responsiveness. In order to investigate the first question, I used prucalopride, another colokinetic which acts on 5-HT4 receptors in the ENS, and found that there was a trend for reduced responsiveness to prucalopride in 6-OHDA rats compared with shams. This suggests there may be a deficiency at the level of the ENS. To address the second question, I peripherally injected 6-OHDA in rats and shown that peripheral injection of 6-OHDA did not induce motor impairments and GI dysfunction in those animals, with no loss of dopaminergic neurons in the nigrostriatal pathway and no change in bead expulsion time. This toxin-induced model is used widely in rats, however the use of 6-OHDA in mice has not been well established. In Chapter 5, I characterised the GI phenotype in the 6-OHDA mouse model. The slowing of bead expulsion from the colorectum and enteric neuron stress exhibited by 6-OHDA rats are also observed in 6-OHDA lesioned mice, providing a clear indication of compromised ENS function. The results suggest there are downstream, trans-synaptic consequences of 6-OHDA lesioning of the MFB. Chronic stress is able to exacerbate motor dysfunction and increase dopaminergic neuron loss in animal models of PD. The aim of Chapter 6 was to investigate whether chronic isolation stress exacerbates GI dysfunction in a genetic, A53T, mouse model of PD. It was shown that A53T mice displayed progressive motor deficits and slowed colonic bead expulsion and whole gut transit, and that chronic stress worsened these PD symptoms. In summary, my studies presented in this thesis have revealed that there are downstream consequences of lesion of the MFB with 6-OHDA in both rats and mice, compromising neuronal functions in the brain, down to the lumbosacral defecation centre and the ENS of the colorectum. Moreover, chronic stress can exacerbate PD-associated GI dysfunction, highlighting the impact of stress on the gut-brain axis in PD.
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    Necessary Steps to Advance Stem Cell-Derived Neural Transplantation Therapy to the Clinic for Stroke
    Law, Kevin ( 2023-04)
    Current treatments for ischaemic stroke are restricted to <10% of patients due to time constraints, leaving many patients untreated. Stem cell-based therapies offer a promising alternate treatment for stroke. Recent advancements in human pluripotent stem cell (hPSC) differentiation protocols to generate defined neuronal populations, such as cortical progenitors, suitable for neural transplantation have enabled preclinical studies to investigate the functional efficacy of this therapeutic approach. Despite evidence that these neural grafts provide neuroprotection and/or replacement of lost neural circuits to reverse motor and cognitive deficits in animal models, there are several outstanding requirements that require attention prior to translation into the clinic. While the FDA may not require animal testing for drug development prior to clinical translation, the testing and advancement of stem cell-based therapies critically relies on models that recapitulate the disease or injury. Although a number of ischaemic stroke models exist in rats, the intracerebral delivery of the potent vasoconstrictor endothelin-1 (ET-1) provides a robust focal ischaemic model, inclusive of a primary insult and vulnerable penumbra capable of being salvaged by stem cell therapy, that is highly reproducible, stable, and relatively quick and easy to induce. However, efforts to achieve a similar model using ET-1 in mice has been underwhelming likely due to insufficient vasoconstriction, thereby hindering the utility of transgenic models that enable probing of key mechanisms, and their use in high-throughput studies for vigorous preclinical testing therapies. In Chapter 3 of this thesis, intracerebral co-administration of ET-1 in the mouse brain with the non-selective nitric oxide synthase inhibitor L-NAME and selective antagonist of vasodilatory endothelin subtype-B receptor RES-701-1 were shown to generate a larger infarct, induce an inflammatory response and exhibit greater motor deficit, as assessed by the ladder walk test in comparison to animals administered with ET-1 alone. Here, we advanced current ET-1 mouse models of ischaemic stroke to facilitate the use of mice for transgenic and/or high-throughput studies. Necessary for clinical translation is the validation that cell products, in this case human pluripotent stem cell (hPSC)-derived neural grafts, generated within current Good Manufacturing Practice (cGMP) clinical conditions behave in a comparable manner to the pre-clinical cell product counterpart. Chapter 4 characterises the differentiation and maturation of a cGMP hPSC line into cortical progenitors in vitro, showing comparable efficacy to generate TBR2+ cortical progenitors as our previously characterised cell lines. Noting the necessity to implant neural progenitors (which engraft with higher cell survival than post-mitotic neurons), we examined the optimal cortical progenitor age for transplantation (day of differentiation 25, 30 and 35) and assessed the impact of a NOTCH signalling inhibitor (aimed at forcing progenitor cell-cycle exit) to circumvent risks associated with neural overgrowths. Assessment of resultant grafts revealed that the treatment of NOTCH inhibitor prior to cell implantation prevented tumour formation. Furthermore, younger cortical progenitors generated a higher proportion of deep layer cortical neurons, and superior and appropriate anatomical innervation of the host brain. We also demonstrated that cryopreserved cGMP hPSC-derived cortical progenitors can generate viable grafts, validating cryopreservation procedures (necessary for clinical translation) for the generation of an ‘off-the-shelf’ cell product for transplantation. This study achieved the first steps towards translating cortical progenitor cell transplantation to repair the ischaemic stroke injured brain. One of the greatest concerns of human pluripotent stem cells in regenerative medicine is the risk of tissue overgrowth/tumours. The incorporation of suicide genes (such as the herpes simplex virus-thymidine kinase gene) into hPSCs can improve the safety of resultant neural grafts by eliminating unwanted cells post-transplant using a systemically delivered prodrug (ganciclovir). A challenge of this approach, however, is the short half-life of the drug in vivo, that is required to coincide with S-phase of the cell cycle (in order to ablate dividing cells). To address this, Chapter 5 utilised a self-assembling peptide hydrogel, with mimetics similar to the brain’s extracellular matrix, to encapsulate the prodrug and consequently prolonged its presentation within the brain. This “one-off” prodrug delivery treatment enabled the efficient activation of the suicide gene in more graft-derived cells, consequently ablating a greater number of proliferative cells within a pluripotent stem cell-derived teratoma in comparison to the standard and suboptimal regime of daily systemic prodrug delivery. Furthermore, within neural grafts, the therapeutic neuronal cells were enriched following our hydrogel-based prodrug delivery. Here, we demonstrated efficient and safe activation of the suicide gene within neural grafts, facilitating the translation of neural transplantation to the clinic. These studies collectively provide important steps in the testing and development of hPSC-derived cortical progenitors and contribute to the advancement of stem cell-based therapies for stroke towards the clinic.
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    Artefact reduction methods for EEG-fMRI and fMRI
    Bullock, Madeleine Frances ( 2023-01)
    Advances in technology have led to increasing use of neuroimaging methods, such as electroencephalography (EEG), magnetoencephalography (MEG) and functional MRI (fMRI), that allow researchers to study brain function in a non-invasive way. However, functional neuroimaging studies are frequently contaminated by noise, or artefact, present in the data, and therefore, methods to remove or minimise artefact are crucial for obtaining accurate, reproducible results. This thesis examines artefact reduction in two different functional neuroimaging modalities - fMRI, and simultaneous EEG-fMRI. Firstly, a systematic review of artefact reduction for EEG-fMRI is presented, which contains two sub-reviews: a review of artefact reduction methods available, and a review of artefact reduction methods used in contemporary studies. The first review successfully distils all published artefact reduction methods from a twenty-year period into clear recommendations for researchers using this imaging modality. The second review found that from EEG-fMRI papers published over a four-year period, most users were selecting one or two similar methods, with up to 15% of users not adequately describing their methods used. A key finding from the work was that hardware-based methods of recording artefact are preferable to data-driven approaches, yet data-driven approaches are most commonly used. The second part of this thesis looks at motion artefact in fMRI studies - specifically, the utility of novel hardware - carbon wire loops (CWL) - for detecting head motion. We aimed to determine whether CWL could detect sub-volume motion onset and if so, whether CWL would suggest further data censoring when using a commonly used data-driven method, Framewise Displacement (FD). We hypothesised that the volume prior to motion onset in FD may often be motion affected, due to motion occurring part-way through volume acquisition, and that this motion would be detected by CWL. The results showed that CWL successfully detects motion onset at a sub-volume (slice-based) level. In addition, CWL detected motion in the volume prior to FD onset in an average of 42% of cases. It was concluded that censoring the volume prior to FD onset should only be done when rigorous motion rejection is necessary and CWL are not available. Together, both these works extend the current knowledge and methods for reducing artefact in neuroimaging studies. The first work provides guidelines for researchers using EEG-fMRI, to reduce artefact and successfully report their methods when publishing. The second work shows a proof of concept that CWL can detect motion in fMRI studies, thus laying the groundwork for more sophisticated motion removal algorithms for fMRI in the future.
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    Investigating the effects of upright positions on cerebral hemodynamics and clinical outcomes in acute ischaemic stroke
    Braico Carvalho, Lilian ( 2023-03)
    The effects of upright positions (sitting and standing) on cerebral haemodynamics early post an ischaemic stroke are not well understood. Neither is their influence on longer term clinical outcomes. Concerns that early upright activity (sitting, standing and walking activities, often termed early mobilisation) may worsen cerebral perfusion within penumbral tissue early post stroke, particularly in people with occlusive disease, guided the development of the research in this thesis. This thesis is comprised of four studies that collectively aimed to shed light on the uncertainty around the effects of head positioning, particularly upright positions, on cerebral haemodynamics and clinical outcomes in acute ischaemic stroke. The first study, a comprehensive systematic review and meta-analysis, highlighted the dearth of studies assessing changes in cerebral haemodynamics in upright sitting and standing in acute ischaemic stroke. Yet getting out of bed, sitting up and walking, are early activity practices that, while part of routine stroke unit care, are not strongly guided by evidence. This review also showed the dearth of head positioning studies that examined occlusive disease as a patient-related factor that may contribute to further reductions in cerebral perfusion during upright activity. This review highlighted important research gaps to be addressed in the subsequent studies. The second study aimed to explore whether occlusive disease contributed to worse clinical outcomes in people with ischaemic stroke treated with very early mobilisation (more frequent out of bed upright activities, started <24h of stroke onset). This post hoc retrospective substudy of the A Very Early Rehabilitation (AVERT) trial showed a greater proportion of participants with occlusive disease having worse 3-month clinical outcomes (modified Rankin Scale, deaths) treated with very early mobilisation compared to usual care. However, no significant associations between occlusive disease, clinical outcomes and mobilisation group were found. Although the study was likely underpowered with small numbers of important clinical events, the potential for occlusive disease to influence outcome warranted further exploration. The third and main study of this thesis, was a prospective study investigating orthostatic changes in cerebral haemodynamics while people with acute ischaemic stroke (within 48h of symptoms onset) moved from lying-flat (0 degrees) to upright (90 degrees) sitting and standing. This was the first study to assess middle cerebral artery mean velocities using transcranial Doppler at upright positions in acute ischaemic stroke at such an early stage (within 48h of stroke onset), and which also included people with occlusive disease. This study also assessed orthostatic changes in mean velocity over time by comparing assessments <48h to a later time (3-7 days post stroke). Finally, this study explored the association between early changes in mean velocities (<48h) to 30-day functional outcome measured with modified Rankin Scale. Results from this study showed significant reductions in mean velocities at upright sitting and standing in people with ischaemic stroke, with and without occlusive disease. Our study also showed no association between orthostatic changes in mean velocity and 30-day functional outcome. Interrupted by the COVID pandemic, a limitation of this study was that it was underpowered to definitively test our hypothesis. Nevertheless, given the relatively large sample, and acute timing compared with previous studies, we believe our results contribute valuable information on the effects of upright postures to cerebral haemodynamics in the first days after stroke. Finally, given the important protective effect of collateral flow to the ischaemic penumbra, the last study aimed to explore the influence of collateral flow on orthostatic changes in mean velocities in acute stroke. The clinical hypothesis was that people with ischaemic stroke with poor collateral flow, would show greater reduction in mean velocities at upright positions compared to those with good collateral flow. Exploratory post hoc analyses were performed on data from perfusion images collected retrospectively and added to the dataset from Study 3. Although the results did not show a significant association between good or poor collateral flow and orthostatic reductions in mean velocity, this study introduced important novel hypotheses related to cerebral haemodynamics and upright activity. In conclusion, this thesis has contributed new evidence and insights into head positioning, upright activity and occlusive disease in acute ischaemic stroke. Mean velocities do reduce at more upright positions in people with acute ischaemic stroke both with and without occlusive disease in the first two days post stroke. However the clinical significance of this finding remains unknown. Future research should focus on better understanding the factors influencing cerebral autoregulation and haemodynamics as well as further assessing patient-related factors (i.e. presence of occlusion or stenosis, collateral circulation) that may influence how cerebral blood flow changes with head position in acute ischaemic stroke.