Florey Department of Neuroscience and Mental Health - Theses
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Genetic and pharmacological targeting of Heat shock protein 72 (Hsp72) in the 5xFAD*Tg30 mouse model of Alzheimer’s disease
The impact of Alzheimer’s disease (AD) is profound. In Australia, around 460,000 patients have dementia, AD being the most common form. An estimated 1.2 million people are involved in their care and it is the second leading cause of death. It is difficult to provide statistics purely on AD due to the difficulty in diagnosis, but an estimated 70% of dementia cases are AD (322,000 patients) (Dementia Australia 2018). Overexpression of the cytoprotective Heat Shock Protein 72 (Hsp72) is currently under investigation as a potential therapeutic option for prevention and treatment of AD due to its many protective mechanisms. The series of projects in this thesis aim to better understand the effects of upregulating Hsp72 in relation to behaviour, cognition and metabolism in a mouse model of AD. The aim of the first study was to replicate and extend on the characterisation of a recently developed mouse model of AD. The mutant 5xFAD (overexpressing human APP and Presenilin-1) was crossed with Tg30 (overexpressing tau) to produce 5xFAD*Tg30 mice and investigate the effects of tau accumulation in the presence of amyloid pathology, which closely resembles human AD. A comprehensive battery of behavioural and physiological tests were performed to observe cognitive and physical decline over time, between 3-8 months of age. In agreement with one previous report (Heraud et.al 2014), we observed a decline in Rotarod performance from 6 months onwards compared with wildtype control (WT). While we noted a decrease in spatial awareness and memory (Morris water maze) and significant genotype differences in anxiety-like behaviour (large open field) by 8 months, memory function (Y-maze) and novel object recognition were not significantly affected. The 5xFAD*Tg30 mice had a 40% decrease in survival by 10 months of age. Additionally, 5xFAD*Tg30 mice were smaller, with a significant difference in body weight, tibialis anterior skeletal muscle weight and tibia length. Our results successfully reproduced aspects of the previously published description of this model (Heraud et.al 2014), while adding further additional characterisation of the model. This initial characterisation study demonstrated that the model develops many aspects associated with AD including frailty, early death and initiation of cognitive decline to complement the previously described AD-like brain pathology, specifically the presence of amyloid-beta pathology with tau accumulation and neurofibrillary tangle (NFT) development, found in this model (Heraud et.al 2014). Hsp72 has been shown to play a cytoprotective role in AD- related research by inhibiting amyloid-beta oligomerization, enhancing its clearance, restoring tau homeostasis and inhibiting neuronal apoptosis. Hence, the aim of the second study was to genetically overexpress Hsp72 and study its effects on the 5xFAD*Tg30 mouse model of AD. As in the first study, we crossed 5xFAD and Tg30 mice, to create the double transgenic, 5xFAD*Tg30, then crossed the double transgenic mice with Hsp72 overexpressing transgenic mice (Hsp72 Tg) to create the triple transgenic, 5xFAD*Tg30*Hsp72Tg. BGP -15 is a compound described to be a co-inducer of Hsp72, and therefore, our third study hypothesized that pharmacologically activating Hsp72 with this compound may be advantageous in preventing or delaying AD progression and associated pathology. 5xFAD*Tg30 mice were treated with BGP-15 or vehicle in their drinking water in a randomised and blinded study in both male and female mice from the ages of 2-10months. A comprehensive battery of behavioural and metabolic tests was conducted for both the second and third studies, and results compared to littermate WT mice. We observed significant declines in Rotarod, Y-Maze and Novel Object performance and increased seizure activity in the 5xFAD*Tg30 mice compared to wildtype, however neither Hsp72 overexpression (study 2) nor BGP-15 treatment (study 3) rescued this decline. Hsp72 overexpression was partially effective in maintaining lean mass in male mice and improved performance on the elevated plus maze. In males there was a 38% decrease in survival rates by 10months of age in the 5xFAD*Tg30 mice, which was improved by BGP-15 treatment to only a 14% loss (p=0.07). In conclusion, BGP-15 may improve survival rates in a gender specific manner (male), while overexpression of Hsp72 leads to maintenance of lean mass in male 5xFAD*Tg30 mice. Both avenues used to target Hsp72 were insufficient, however, to protect against the observed cognitive deficits in the model.
Functional and structural brain networks during epileptic spikes in human brain
Accurate localisation of the brain region responsible for the seizure onset is a critical step for refractory epilepsy patients to gain access to surgical treatment. In this work, EEG, functional MRI and diffusion-weighted MRI were used to enhance the current understanding of functional and structural properties of brain networks involved in epileptic activity in patients with refractory focal epilepsy. The findings of the studies included in this thesis support the view that epilepsy is a network disorder, and epileptic activity is organised in a complex network. This work showed that results from simultaneous EEG-fMRI studies can have a critical impact on patient management, including influencing the decision for targeted surgical resection, with good subsequent seizure outcomes. Preforming EEG-fMRI studies in patients with frequent spikes during inpatient video-EEG monitoring, while on reduced or withdrawn antiepileptic medications, may lead to a much higher proportion of successful cases. Similarly, automatic spike detection method can improve detection of epileptic spikes in scalp EEG recordings, significantly shorten the analysis time, and enhance the statistical power of EEG-fMRI analysis, revealing additional elements of the patient’s epileptic networks. This work proved that the addition of spike variability information into standard fMRI analysis of the EEG-fMRI data allows for better localisation of the origin of epileptic activity and its spread across the network. In addition, investigating the structural connections that may underlie functional networks can enhance the current understanding of the propagation pathways used by epileptic activity. This work demonstrated the utility of advanced analysis methods in the investigation of refractory epilepsy. Advanced neuroimaging techniques can better localise the epileptic onset and propagation of epileptic activity. Combination of information from different diagnostic modalities bridges the gap between neurophysiology (EEG) and imaging (MRI) of epilepsy and may be a useful adjunct to the presurgical assessment of refractory epilepsy.
Targeting HCN4 channels in epilepsy
Epilepsy is a prevalent neurological disorder that affects around 65 million people worldwide. Despite optimal treatment with modern antiepileptic drugs, about one third of patients will continue to have seizures, along with undesirable side effects. Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels are encoded by four genes (HCN1-4). HCN channels have four isoforms (HCN1-4) and produce HCN-mediated currents (Ih) that exhibit pacemaker properties critical for regulating the hyperexcitable neuronal activity seen during seizures. This thesis explores the impact of both pharmacological and molecular HCN channel block on seizure susceptibility and neuronal excitability. Broad-spectrum HCN channel block using ivabradine significantly reduced the seizure susceptibility of wildtype and Scn1a Dravet mice in two proconvulsant assays. Testing isoform-selective HCN channel blockers, the HCN2/1-preferring channel blocker, MEL55A, increased seizure susceptibility. Whereas, the HCN1-preferring channel blocker, MEL57A, had no effect on seizure susceptibility. The HCN4-preferring channel blocker, EC18, significantly reduced seizure susceptibility in two proconvulsant assays in vivo while displaying a safe drug profile. Furthermore, the conditional knockout of HCN4 channels in adult mice was also sufficient to significantly reduce seizure susceptibility in proconvulsant tests with minimal behavioural effects. Interestingly, EC18 showed no effect on seizure susceptibility when administered intraperitoneally to the conditional HCN4 knockout mouse model indicating seizure protection is HCN4-dependant. Moreover, electrophysiological as well as multi-electrode array (MEA) recordings indicated a significant reduction in parameters relating to neuronal excitability after treatment with the HCN4-preferring channel blocker, EC18. Together these results indicate that HCN4 channels are important mediators of neuronal network excitability suggesting they are promising anti-seizure drug targets with minimal adverse effects.
Early Mobilisation After Stroke: Barriers and Enablers for Effective Use of Population-Level Evidence to Inform Individualised Clinical Decision-Making
Early mobilisation, defined as sitting out of bed, standing or walking early after stroke, is an important constituent of acute stroke unit care. However, the multifaceted and complex nature of early mobilisation evidence and interventions, clinical practice guideline development and reporting, and the characteristics of stroke patients and their outcomes challenges individualised decision-making. The overall objective of this thesis was to investigate the barriers and enablers in the effective use of population-level evidence to inform individual patient-level clinical decision-making for early mobilisation post-stroke. The objective was achieved by conducting five studies: (1) a review of early mobilisation clinical practice guidelines as decision-support tools for individual patient-level decision-making; (2) a meta-analysis of individual participant data from EM trials, (3) an investigation of factors guiding early mobilisation decision-making by expert stroke clinicians, and investigation of how well (4) the utility-weighted modified Rankin scale, and (5) modified Rankin scale reflect post-stroke burden for an individual patient. (1) Based on the findings from the review of clinical practice guidelines, the decision-support requirements were met to a varying degree by early mobilisation clinical practice guidelines. Four key recommendations were formed for the future development of clinical practice guidelines. These included more granular descriptions of patient and stroke characteristics to allow tailoring of decisions to individual patients; clarity about when clinical flexibility is appropriate; a detailed description of the intervention dose, and physical assessment criteria including safety parameters. (2) The individual participant meta-analysis allowed the inclusion of individual patient-specific information and further strengthened earlier evidence from conventional meta-analyses that commencement of early mobilisation should only be considered after 24 hours post-stroke. It also reinforced the importance of adequate reporting of early mobilisation interventions to ensure the applicability of evidence to individual patients. (3) Interviews with expert stroke clinicians revealed that more than 80 percent of stroke experts considered stroke type and severity, medical stability to be the most important factors contributing to decision-making about early mobilisation. Inadequate staffing, equipment and low level of staff expertise were barriers for early mobilisation. (4) Based on the investigations of the utility-weighted modified Rankin scale, high variability in individual patient-centred utility values between and within mRS categories, over time post-stroke, and using different derivation methods was found. This variability is not adequately reflected in the utility-weighted modified Rankin scale. (5) Quality of life and activities of daily living domains demonstrated patient-specific patterns of post-stroke burden across the mRS and UW-mRS. From this research, gaps in the early mobilisation evidence base were identified that require future exploration of existing data and the development of new clinical trials to better support evidence-based clinical decision-making. Detailed areas of improvement for clinical practice guidelines as decision support tools were also identified to effectively translate population-level evidence to inform complex clinical decision-making at an individual patient-level. Finally, the identified multifaceted patterns of post-stroke burden across the utility-weighted modified Rankin scale and modified Rankin scale may facilitate appropriate assessment, articulation and interpretation of the outcomes for individual patient decision-making. The collective work has substantially contributed to systematically identifying the barriers and enablers in using population-level evidence to inform patient-level clinical decision-making in this challenging clinical context.
Characterization of white matter asymmetries in the healthy human brain using Diffusion MRI fixel-based analysis
Magnetic resonance imaging (MRI) has revolutionized the way to investigate brain structural connectivity non-invasively. Diffusion MRI can be used to obtain local estimates of the white matter fibre orientations in the brain, which in turn can be used to study changes in the local fibre specific properties and/or in conjunction with fiber-tracking algorithm to reconstruct a representation of the white matter pathways in the brain. In recent years, the Diffusion Tensor model has played an important role in modelling the diffusion of water within white matter bundles. Diffusion tensor derived metrics such as fractional anisotropy (FA) have been used extensively for investigating white matter using approaches such as voxel-based analysis. One of the limitations of the diffusion tensor model is that it is not capable of appropriately modelling regions that have complex fibre architecture (such as crossing fibres). This makes tensor-derived measures unreliable measures to assess the white matter. Recent contributions toward the study of brain asymmetry have suggested asymmetry of brain anatomy and function are observed in the temporal, frontal, and parietal lobes. Several studies have used diffusion tensor model to study asymmetry in various regions of the human brain white matter. However, given the limitations of the tensor model, the nature of any underlying asymmetries remains uncertain. This research aims to provide to provide a more robust characterization of structural white matter asymmetries than those previously derived using the tensor model, by using quantitative measures derived from the spherical deconvolution model, and a whole-brain data-driven statistical inference framework such as Fixel-Based Analysis, that is both sensitive and specific to crossing fibres; we furthermore apply this approach to a state-of-the-art publicly available diffusion MRI dataset.
Developing peptidomimetic ligands for relaxin family peptide receptors
Relaxin family peptides that target G protein-coupled receptors (GPCRs), have important therapeutic applications (e.g., heart failure, fibrosis, cancer, and disorders related to psychiatry, drug addiction, eating, or colon motility). However, these family peptides have an insulin-like complex heterodimeric structure that is difficult to produce and modify to improve their pharmacokinetic properties (e.g., half-life). I have carried out structure-activity relationship (SAR) studies on these peptides leading to the generation of simplified agonists and/antagonists for the GPCRs, RXFP1, RXFP3, and RXFP4. These novel peptides are easier to prepare in larger yields and retain high affinity for their receptors. They are important research tools, and potential drug leads for the treatment of human diseases as mentioned above. Chapter 1 summarises the literature on the SAR studies of H2 relaxin, H3 relaxin and human INSL5 leading to the design and development of simplified agonist and antagonist. Chapter 2 describes all the materials and methods used in this study. Chapter 3 summarises the SAR studies on H2 relaxin. H2 relaxin is known to have strong anti-fibrotic, vasoprotective, angiogenic, and vasodilatory effects. The receptor for H2 relaxin, RXFP1, is a potential target for the treatment of heart failure, fibrosis and related disorders, including liver cirrhosis, and preeclampsia. Our laboratory has designed a B-chain-only analogue, B7-33, which was shown to be a potent RXFP1 agonist in cells endogenously expressing RXFP1 and in several animal models of fibrosis (mice vs rats). B7-33 is 27 residues long and has short circulation time in serum (t1/2= ~6 min). I have shown that B7-33 can be further truncated up to 4 residues, and it’s in vitro half-life in human serum can be increased from 6 minutes to 60 minutes. I also have developed a B7-33-based single-chain RXFP1 antagonist for the first time. These analogues are important research tools, and drug leads for the treatment of human diseases (e.g. fibrosis-related diseases, prostate, and other cancers). Chapter 4 summarises the SAR studies on the neuropeptide H3 relaxin. The receptor for the H3 relaxin hormone, RXFP3, is an attractive pharmacological target for the control of eating, addictive, and psychiatric behaviours. This chapter has two parts. In the first part, I have developed four novel analogues based on two chains, and two disulfides bonded H3 relaxin analogue, also known as A2 analogue. I have introduced amide, alcohol, carbamate, and ester functionality at the C-terminus of the B-chain of A2 analogue. In terms of binding to RXFP3, the carboxylic acid remains to be the most suitable functionality. However, the cAMP response seems to be better with the C-terminal ester function. Such results highlight the possibility of generating drug candidates, as applied to H3 relaxin, to slow down the release or generating a long-acting analogue of H3 relaxin. In the second part, I have developed an alpha-helix stabilising chemical method. Using this method, I have produced a high yielding single-chain RXFP3 agonist and antagonist. The agonist peptide, H3B10-27-13/17alphaF, is 20-fold more stable in vitro in human serum compared with the control peptides (H3B10-27-13/17EA and H3B10-27-13/17FF). The novel RXFP3 antagonist, H3B10-22R-13/17alphaF, is only 14 amino acid long compared with the previously reported 23 residues antagonist. These analogues will facilitate the identification of physiological roles of RXFP3 and drug leads for the treatment of neurological disorders such as stress and anxiety. Chapter 5 summarises the SAR of insulin-like peptide 5 (INSL5), a gut hormone. Its receptor RXFP4 is a potential target for the treatment of anorexia, obesity, and colon motility disorders (e.g. constipation and diarrhoea). Our laboratory was the first to design and develop an RXFP4-specific agonist peptide known as analogue 13. However, there was no RXFP4-specific antagonist reported in the literature. The focus of this study was to utilise the non-specific RXFP3/RXFP4 antagonist, deltaR3/I5, as a template to design an RXFP4 specific antagonist rationally. Based on analogue deltaR3/I5, I have developed an analogue 17, which is RXFP4-selective. Analogue 17 is an ideal template for further development into a specific high-affinity RXFP4 antagonist as tool and drug leads. Chapter 6 summarises the findings in this study and provides future directions.
The Role of MERTK in Central Inflammatory Demyelination
Multiple sclerosis (MS) is, at least in part, an autoimmune demyelinating disease of the central nervous system. It is the most common cause of neurological dysfunction in young adults. In addition to demyelination, autoimmune destruction in MS results in oligodendrocyte loss, local inflammation and neurodegeneration. Later in the disease course, a pattern of neurodegeneration may emerge without obvious evidence of adaptive immune activation. Current evidence implicates dysregulation of the innate immune system as a major contributor to the neurodegeneration of progressive MS. It is well established that innate immunity influences central nervous system damage and modulates myelin repair. The TAMs (TYRO3, AXL and MERTK) are a family of receptor tyrosine kinases expressed by discrete innate immune cell types, including macrophages, microglia and dendritic cells. They are involved the homeostatic regulation of adult, fully differentiated tissues that are subject to constant intrinsic and environmental challenge. The primary aim of this thesis was to provide additional insight into the biology of the MERTK receptor by examining its role in commonly employed mouse models of MS. In addition, I sought to identify whether serum levels of MERTK correlated with disease activity in MS patients experiencing relapse. I established that heterozygote deletion of Mertk in CD11+ve cells worsens the clinical severity of EAE in male mice, demonstrating for the first time that changes in the expression of a TAM receptor can alter the outcomes of a model of MS. Conversely, homozygous deletion did not result in EAE exacerbation. Heterozygote deletion of Mertk in CD11+ve cells does not alter the outcomes of cuprizone-induced demyelination, suggesting that CNS-resident CD11c+ve cells (microglia) are not implicated in the clinical exacerbation observed in EAE cohorts. In human subjects, MERTK expression by monocytes and dendritic cells does not correlate with periods of increased inflammatory activity in MS. MERTK expression by circulating dendritic cells is very limited. Taken together, the data in this thesis offer compelling evidence that MERTK is an important regulator of the outcomes of central inflammatory demyelination. They justify further research efforts in this area to better understand the mechanism of these disease states and to develop therapeutic targets for the treatment of MS.
Sleep-wake dysfunction in human ischaemic stroke
Sleep-wake dysfunction is increasingly recognised as a key modifiable risk factor and consequence of stroke. Chronic sleep-wake abnormalities, characterised by excessively long sleep duration or sleep disorders, increase the risk of ischaemic stroke. Following stroke, de novo sleep-wake impairment is common and associated with poor recovery. However, the pathogenesis and evolution of sleep-wake disturbances in stroke have been poorly characterised thus far, largely due to methodological limitations. In this thesis, gold-standard sleep measurement tools and advanced MRI methodologies were applied to investigate the impact of chronic stroke on sleep-wake function. There were three primary research questions for this thesis, and each formed the conceptual framework for three major studies: (1) To what extent is sleep-wake dysfunction associated with both stroke risk and post-stroke evolution? (2) What are the neurodegenerative markers of sleep-wake after stroke? (3) What are the sleep architectural and sleep-respiratory characteristics of chronic stroke patients relative to healthy controls? To address the first question, a scoping systematic review of over 5,000 studies was conducted in order to assess the bidirectional relationship between sleep and circadian rhythm dysfunction in human ischaemic stroke. A qualitative synthesis of the extant literature showed that excessively long sleep duration and sleep disorders significantly increase the risk of ischaemic stroke. On the other hand, acute stroke patients exhibit fragmented sleep architecture in the weeks following the incident event – potentially driven by newfound sleep disorders which may also be associated with post-stroke topography and recovery. These findings support a bidirectional relationship between sleep-wake dysfunction and ischaemic stroke with important clinical implications. To expand on limitations of prior studies identified in the aforementioned systematic review, the associations between regional neurodegeneration and objectively measured sleep were investigated in a cohort of mild-to-moderate stroke patients and healthy controls from the Cognition and Neocortical Volume After Stroke (CANVAS) study. Stroke patients with excessively long sleep duration and poor sleep efficiency exhibited volumetric reductions to the thalamus and amygdala relative to healthy controls. Next, a novel method known as a whole brain fixel-based analysis was utilised to investigate fibre-specific white matter degeneration in stroke patients with poor sleep. Stroke patients with excessively long sleep duration exhibited tract-specific neurodegeneration to the cortico-ponto-cerebellar tract. These findings suggest that poor sleep efficiency or long sleep duration may contribute to neurodegeneration following stroke. The final study in this thesis aimed to characterise hemispheric sleep architecture and sleep-respiratory characteristics in stroke patients >4 years after their incident event using gold-standard polysomnography. In a subsample of patients from the CANVAS study, stroke patients and matched controls underwent overnight ambulatory polysomnography and completed an array of sleep and circadian questionnaires. Over half of all stroke patients in this sample exhibited undiagnosed moderate to severe obstructive sleep apnoea. Stroke patients had nearly 40% less restorative slow-wave sleep and potentially compensatory increases in lighter sleep stages relative to healthy controls. Sleep architectural disturbances were not attenuated by obstructive sleep apnoea. There were no sleep architectural differences in the stroke-affected versus healthy-hemisphere. These findings suggest that sleep impairment post-stroke is unlikely to be driven by comorbid obstructive sleep apnoea or the hemispheric distribution of stroke lesions. Furthermore, these results highlight the importance of formal sleep studies in stroke patients in order to identify undiagnosed obstructive sleep apnoea and fragmented sleep architecture. The overall findings from this thesis offer valuable insight into the potential in vivo pathogenesis of sleep-wake dysfunction after stroke and the evolution of sleep abnormalities in the chronic stages of stroke. The clinical-pathogenic implications of sleep-wake dysfunction in stroke are unravelled, and a research agenda for future studies in this emerging field of medicine is outlined.
The modulation of cortical dendrites during sensory perception and learning
Sensory perception arises in the cortex by integrating external information from the environment with internal representations and the current brain state. This process is supported by the structure of the neocortex and the organisation of excitatory inputs onto its core computational element: the pyramidal neuron. In layer 2/3 pyramidal neurons, external (feedforward) information mainly target the somatic region, while internal (feedback) information runs through layer 1 and lands onto distal tuft dendrites. Cortical dendrites have active properties that could be important for sensory processing and could provide a cellular mechanism to support the flexibility of sensory representations during learning. In this thesis, I addressed the role of L2/3 pyramidal neuron dendrites during sensory perception and learning in three parallel studies that investigated: 1) how inputs from another sensory area affect early stages of sensory processing; 2) the modulation of sensory processing in dendrites of the auditory cortex following fear conditioning; 3) changes in dendritic activity during perceptual learning of an auditory discrimination task. To tackle these questions, a combination of two photon Ca2+ imaging and whole cell patch clamp electrophysiology in vivo, together with behavioral testing and optogenetics manipulation, was used. The results presented here confirm that dendrites encode sensory information and show that they can undergo plastic changes during learning. The findings also illustrate the existence of compartmentalised activity in L2/3 pyramidal neurons and of a cellular mechanism for the control of action potential generation that involve dendritic integration. The results presented in this thesis highlight the importance of the upper layers of the cortex for flexible sensory representation through the integration of feedback and feedforward information.
Remyelination in the aging brain and the effects of growth factor supplementation on oligodendrogenesis
In multiple sclerosis (MS), chronic demyelination initiated by immune-mediated destruction of myelin, leads to axonal damage and neuronal cell death, resulting in a progressive decline in neurological function. The development of interventions that potentiate remyelination could hold promise as a novel treatment strategy for MS. Seminal work in the Merson group has demonstrated that neural precursor cells (NPCs) residing in the subventricular zone (SVZ) of the adult mouse brain contribute significantly to remyelination in response to CNS demyelination and can regenerate myelin of normal thickness. However, aging takes its toll on the regenerative potential of NPCs and reduces their contribution to remyelination. In this study, I investigated how aging could affect the NPCs contribution to oligodendrogenesis during the remyelination process and whether the delivery of growth factors into the brains of aged mice could potentiate the oligodendrogenic potential of NPCs. To map the fate of NPCs in response to demyelination induced at different postnatal ages, Nestin-CreERT2; Rosa26-LSL-eYFP mice were gavaged with tamoxifen at either 8 weeks, 30 weeks or one year of age before being challenged with cuprizone for a period of six weeks. Using osmotic minipumps, I infused epidermal growth factor (EGF) and/or heparin-binding EGF-like growth factor (HB-EGF) or artificial cerebrospinal fluid (vehicle) into the cisterna magna for a period of two weeks beginning at the peak of cuprizone-induced demyelination (n=6-8 mice per group). Mice were perfused six weeks after cuprizone withdrawal and the contribution of NPCs to oligodendrocyte regeneration in the corpus callosum was assessed. Our data reveal that although NPC-derived oligodendrocyte generation declined dramatically with aging, this decline was at least partially reversed by growth factor infusion. Notably, co-infusion of EGF and HB-EGF increased oligodendrocyte regeneration by two-fold in some regions of the corpus callosum. The results of this study demonstrate the beneficial effects of EGF and HB-EGF for increasing the contribution of NPCs to oligodendrogenesis during the remyelination process and indicate that modulation of progenitor responses could hold therapeutic potential for combating the negative effects of aging upon remyelination efficacy.
Age related dopamine 1 and dopamine 2 receptor expression in addiction-related behaviours
Methamphetamine (meth) is a significant social and public health concern worldwide, and a growing problem in Australia. One factor contributing to meth use disorder is the lasting memory of its rewarding experience, which can lead to persistent use in vulnerable individuals. Epidemiological data show that adolescence is a period of heightened vulnerability for developing meth use disorder. Furthermore, sex differences exist in numerous aspects of meth use motivations, behaviours, and consequences. Despite this, few studies have investigated age and sex effects of meth on brain and behaviour. A unified neural mechanism by which substances of abuse, including meth, produce their addictive properties is by increasing dopaminergic transmission throughout the mesocorticolimbic dopamine system. Although dopamine binds to and activates several subclasses of receptor in brain regions implicated in reward processing, the dopamine receptors 1 (D1) and 2 (D2) have been reported to be particularly important for drug-affected behaviour. Importantly, levels of D1 and D2 have been shown to fluctuate throughout development. D1 and D2 signalling may therefore be important mediators of adolescent vulnerability to substance use. However, there are numerous inconsistencies in the literature that describe developmental changes in D1 and D2 expression. Systematic characterisation of these changes is therefore critical for a full understanding of how changes to the dopamine system may affect susceptibility to meth (and other substance) use disorder. As such, the first aim of my thesis was to investigate the postnatal developmental trajectory of D1 and D2 in addiction-related brain regions. This was achieved using D1- and D2-green fluorescent protein (GFP) transgenic mice, starting from the juvenile period through to adulthood. The results showed region-specific changes in D1 and D2 expression occur across development, with the insular cortex (insula) showing the most dramatic changes. In particular, the density of D1 compared to D2 expressing neurons (D1:D2 ratio) in the insula substantially increased from adolescence to adulthood in males. Since substance use disorders are male dominant disorders which often have an adolescent onset, this reduced D1:D2 ratio may be relevant in understanding the neurobiological basis of substance use disorders. The second aim of my thesis was to investigate the role of insula D1 and D2 in potential age and sex differences in meth conditioned place preference (CPP) and aversion (CPA). Although no age or sex differences were observed in CPP to 0.1 or 3 mg/kg meth at a group level, analysis of individual data revealed females were less likely to form a place aversion compared to males, and adolescents formed more of a place preference to 0.1 mg/kg meth compared to adults. Conditioning with 3 mg/kg meth led to age differences in insula D1:D2 ratio in males, reduced age differences in insula D1:D2 ratio in females, and increased the activation of insula D2 expressing cells in adults compared to adolescents. Insula activity and expression of D1 and D2 did not correlate with place preference behaviour. Taken together, these findings suggest distinct sex differences in D1 and D2 expression across development in addiction-related cortical and striatal brain regions may underly age- and sex-associated vulnerability to meth-related behaviours.
Space for recovery after stroke: Exploring the role of the physical environment in inpatient rehabilitation facilities
The aim of this thesis was to explore the role of the physical environment of inpatient rehabilitation facilities in stroke recovery. The purpose of rehabilitation is to help stroke survivors to re-learn skills and abilities lost as a result of stroke, or to learn new skills to adapt to their changed condition. Research in other healthcare environments suggests that hospital design can impact patient outcomes, but there is little evidence specific to rehabilitation. This thesis embraces the complexity of rehabilitation environments and explores the physical environment as an essential and integrated component of this complex system. This exploration began with a scoping survey to identify and describe all inpatient rehabilitation facilities in Victoria, Australia. This survey revealed 64 facilities, most of which had not been purpose-built for rehabilitation. Rehabilitation facility design appears to be influenced by evidence from acute medical settings and current design trends, rather than reflecting the unique purpose of rehabilitation. A series of expert elicitation workshops were then conducted to define – for the first time – what is important in the physical environment of inpatient stroke rehabilitation facilities. Thirty experts participated, including policy makers, researchers and designers in learning and healthcare environments, clinical staff, and patients. A Value-Focused Thinking methodology was used to facilitate the workshops. The experts defined 16 criteria thought to be fundamentally important (including efficiency, patient practice, activity and rest, emotional well-being, and safety), and 14 criteria that could be a means to achieving these fundamentally important things. Together, these criteria comprise a framework which can be used to guide research and design in this complex area. This framework informed a multiple-case study in two stroke inpatient rehabilitation facilities. Convergent mixed-methods were used to produce a rich and thorough exploration of the cases. Twenty inpatients from Case 1 participated, and 16 from Case 2. The physical environment was described using field notes, photographs, floor plans, and checklists. Walk-through semi-structured interviews were used to explore patients’ experience of the physical environment. Systematic observation (behavioural mapping) and questionnaires were used to investigate patients’ behaviour and emotional well-being in the environment, and a retrospective audit of patient falls was conducted to investigate patient safety. Four interrelated themes described the patient experience: 1) entrapment and escape; 2) power, dependency, and identity in an institutional environment; 3) the rehabilitation facility is a shared space; and 4) the environment should be legible and patient-centred. Quantitative data revealed that patients spent over 75% of their time in their bedrooms. Comparison between cases suggested that the physical environment played a role in patients’ behaviour, emotional well-being, and safety. Qualitative and quantitative findings were then merged using joint display tables and narrative integration. This robust analytic process produced a new conceptual model of the role of the physical environment in stroke patients’ behaviour, emotional well-being, and safety in rehabilitation, emphasising the importance of variety and interest in the environment, privacy without isolation, and patient-centred design. The findings from this study provide meaningful direction for rethinking rehabilitation facilities and guiding real-world health design practice.