Centre for Neuroscience - Theses
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Tau and beta-amyloid deposition, structural integrity, and cognitive function following traumatic brain injury in Australian war veterans
Background: Traumatic brain injury (TBI), has been diagnosed in over 355,000 US military service personnel since 2000. Epidemiological research indicates that veterans with TBI are two-to-four times more likely to develop dementia than controls; however, mechanisms contributing to this relationship are poorly understood. The aim of this study was to investigate if Vietnam war veterans with a TBI show evidence of Alzheimer’s disease (AD) pathological markers, as assessed by A-amyloid, tau and glucose metabolism using PET, as well as structural MRI, including diffusion tensor imaging, and neuropsychological testing. Methods: Sixty-nine male veterans - 40 with TBI (aged 68.0±2.5 years) and 29 controls (aged 70.1±5.3 years) - underwent A-amyloid (18F-Florbetaben), tau (18F-AV1451) and 18F-FDG PET, MRI, psychiatric and neuropsychological assessment. The TBI cohort included 15 participants with mild, 16 with moderate, and 9 with severe injury. Fractional Anisotropy (FA) was employed as a measure of white matter tissue integrity. PET Standardized Uptake Value Ratios (SUVR) were calculated using the cerebellar cortex as reference region. Analyses were adjusted for IQ, age, ApoE status and psychiatric comorbidities. Results: Veterans with moderate-to-severe TBI performed significantly worse than controls on composite measures of memory and learning (M = -0.55 0.69, t(67) = 2.86, p=0.006, d=0.70) and attention and processing speed (M = -0.71 1.08, t(52) = 2.53, p=0.014, d=0.69). The moderate-to-severe TBI group had significantly lower FA than controls in the genu (F(3,36)=8.81, p<0.05, partial 2 = 0.17), and body (F(3,36)=4.39, p <0.05, partial 2=0.14) of the corpus callosum, as well as in global white matter (F(3,36)=5.35, p <0.05, partial 2=0.13). There were no significant differences in 18F-Florbetaben or 18F-AV1451 uptake amongst the groups, however the moderate-to-severe TBI group had significantly lower 18F-FDG retention than controls in the mesial temporal region (F(8,44) = 2.21, p <0.05, partial 2 = 0.13). No differences were found between the mTBI group and controls on any of the outcome measures. Conclusions: These findings indicate that moderate-to-severe TBI, but not mTBI, is associated with later-life cognitive deficits, and diminished global white matter integrity, specifically in the corpus callosum. However, these deficits are not associated with AD pathology. These results are consistent with current evidence of white matter axonal damage as the primary source of cognitive impairment in TBI, and are not reflective of a neurodegenerative process.
Investigating cortical oscillations, coherence and seizure susceptibility in a mouse model of autism
Autism spectrum disorder (ASD) is a pervasive neurodevelopmental disorder diagnosed by difficulties in social communication, repetitive behaviour and/or restricted interests. A high proportion of ASD patients also experience seizures and abnormal brain activity as recorded via electroencephalography, however the underlying biological mechanisms of increased seizure susceptibility in ASD are unknown. Neuroligin-3 (NL3) is a neuronal adhesion protein involved in regulating synaptic structure and function. A rare point mutation at position 451 of the Neuroligin-3 amino acid sequence converts an arginine to a cysteine residue is associated with ASD and reduces NL3 protein levels by 90%. This NL3 R451C mutation was identified in two Swedish brothers with ASD, one of whom was diagnosed with comorbid epilepsy. The mutation is replicated in NL3R451C mice that exhibit reduced preference for social interactions, increased repetitive behaviours, and increased performance in memory and some motor testing. NL3R451C mice also display region-specific differences in inhibitory and excitatory neurotransmission in brain slices increased dendritic complexity, and reduced number of PV-inhibitory interneurons, all of which could contribute to changes in seizure susceptibility and oscillatory activity. Several animal models of ASD show spontaneous seizures and increased susceptibility to experimentally induced seizures, however whether NL3R451C mice have altered seizure susceptibility is unknown. It is well established that brain neuronal activity generates electrical rhythms that underlie cognitive phenomena in humans and may be altered in ASD. Gamma oscillations in the 30-100Hz range are frequently increased with demanding cognitive load and are thought to orchestrate spatially disparate cortical regions. Several clinical studies have pointed to alterations in gamma oscillations and in the coherence of oscillatory activity measured between two disparate cortical regions in ASD patients. To determine whether gamma oscillations are altered in NL3R451C mice, the glutamatergic NMDA receptor antagonist ketamine (20mg/kg; a potent inducer of gamma oscillations) was administered to adult male and female NL3R451C and WT mice and oscillatory activity recorded via electrocorticography (EcOG). PV-interneurons have been experimentally isolated to reveal they are the major drivers of gamma rhythms following ketamine administration. Baseline oscillatory activity from 6 and 10 week old male mice was examined as well as interhemispheric coherence in male and female NL3 mutants and wild type littermates. Gamma oscillatory power in male wild type and NL3R451C mice was comparable following administration of saline and was enhanced to a similar degree following ketamine (20mg/kg) administration. Non-gamma oscillations were also unchanged by ketamine administration. Interhemispheric coherence, however, was significantly higher in 6- week old male NL3R451C compared to control mice for both beta and gamma oscillation ranges. In 10-week old male mutants, there was a significant effect detected for theta, alpha and beta oscillations. Because female mice carry two copies of the X-linked NL3 gene it was hypothesised that any oscillatory activity effects may be exacerbated by the R451C mutation. Gamma band oscillations following administration of ketamine (20 mg/kg) were statistically not significant when WT, heterozygous and homozygous females were compared. Additionally, no statistically significant differences were found for interhemispheric coherence in females. To gain further understanding of the role of the NL3 R451C mutation in epilepsy, seizure susceptibility was investigated in NL3R451C male mice using pentalenetetrazole (PTZ) at both low (20 and 30 mg/kg) and high (50mg/kg) doses. Administration of high dose PTZ is associated with behavioural changes culminating to convulsive seizures. The Racine behavioural scale was used to quantify behavioural seizure severity over time in response to high dose PTZ. In mice, low dose PTZ results spike-wave discharges (SWDs) and a loss of responsiveness resembling absence seizures in patients. Electroencephalography (EcOG) recordings of neuronal activity at the cortical surface was utilised to detect SWDs following administration of low dose PTZ over a 30 minute period. NL3R451C mice showed a strong trend for shorter SWD duration although EcOG analyses of SWD frequency and duration showed similar susceptibility in both mutants and WT littermates to low dose PTZ-induced seizures. Following administration of high-dose PTZ (50mg/kg), NL3R451C mutants were slower to progress to severe seizure scores and overall progressed to milder seizures on the racine scale over the 30-minute testing period. The results of the present study show reduced seizure susceptibility and increased coherence in male NL3R451C mice. These changes might reflect underlying structural and neurochemical alterations present in NL3R451C mice. The reported increase in cortical inhibitory neurotransmission in NL3R451C mice may underlie the resistance to PTZ-induced seizures identified in this study. Similarities in gamma oscillations may indicate similar functioning of NMDA receptors and the PV-interneurons that are strongly implicated in the generation of gamma rhythms. Higher interhemispheric coherence in male NL3R451C mice could stem from differences in cognitive, behavioural and cortical changes present in this model. Further research is needed to clarify relationships between behaviour and oscillatory activity in NL3R451C mice.
Rehabilitation management of post-stroke spasticity
Spasticity is a common manifestation of a neurological condition such as stroke and contributes to long-term disability. Spasticity is disordered sensorimotor control that results from damage to upper motor neurons and presents as involuntary activation of muscles that can be intermittent or sustained. The resulting limb deformity and abnormal positioning cause a diverse range of patient-centred problems such as pain; difficulty walking, transferring, feeding or dressing; and can make it difficult for a caregiver to clean the palm or apply a splint. Given the complexity of spasticity related issues, expert opinion is that spasticity management should entail a multidisciplinary rehabilitation program that may be complemented with botulinum toxin treatment. However, there is a paucity of evidence for the efficacy of rehabilitation programs for spasticity management. In this thesis, four studies address current gaps in evidence-based practice. Study 1 is a systematic review of the effectiveness of multidisciplinary rehabilitation following botulinum toxin and other intramuscular treatments for post-stroke spasticity. Despite low-quality evidence that multidisciplinary care lessens impairment and improves active function in the upper limb, the types and intensities of rehabilitation programs that improve patient-centred outcomes remain unknown. No trials to date have explored the effect of multidisciplinary rehabilitation on passive function, caregiver burden, or the individual’s priority goals for treatment. This study identifies gaps in current research relating to methodological rigour, appropriate study designs and meaningful outcome evaluation. Study 2 compares the effectiveness of a high-intensity ambulatory rehabilitation program and a usual care, low-intensity therapy program in improving patient-centred outcomes after botulinum toxin type A treatment. Goal achievement and satisfaction benefits persisted beyond the duration of spasticity reduction in both groups. While patient-centred outcomes were not influenced by the intensity of the ambulatory rehabilitation program, high-intensity therapy was associated with greater upper limb goal attainment. This suggests that intensive therapy may modify the black box of rehabilitation. Further research is required to evaluate this effect and determine which elements of therapy programs optimise outcomes. Study 3 demonstrates the use of a stroke rehabilitation taxonomy to describe the activities and interventions that therapists used during rehabilitation programs. The relationships between rehabilitation activities prescribed and treatment goals and limbs injected are also examined. Study 4 explores the key implementation processes that may affect the outcome of the high-intensity rehabilitation programs compared with usual care. The influence of program adaptations such as provision of group rather than individual sessions, contextual or organisational differences between rehabilitation centres, therapists’ experiences with managing patients following BoNT-A injections and the effect of goal-directed therapy on outcomes needs further evaluation. This work was undertaken to test the hypothesis that multidisciplinary rehabilitation programs following botulinum toxin type A injections for post-stroke spasticity improve patient-centred outcomes. The study findings guide the recommendations for future research and clinical practice that can be used to improve service delivery and the evidence base for rehabilitation interventions after treatment with botulinum toxin type A for post-stroke spasticity.
The role of steroid hormones in schizophrenia: unravelling the mechanism of 17-beta estradiol and raloxifene on cognitive function
The glycoprotein reelin is integral for brain development and maintaining synaptic plasticity, and its expression is reduced in schizophrenia brains. Whether stress, a risk factor for schizophrenia, is mediated by altered reelin levels is unknown. Here, we showed that adolescent treatment with corticosterone (CORT), the major stress hormone, in a reelin heterozygous mouse model, induced hippocampal-dependent cognitive deficits in both male and female mice. Interestingly, female mice showed sex-specific molecular changes in the dorsal hippocampus after CORT treatment. This suggests a significant role of estrogens in mediating stress responses and cognition. One mechanism through which estrogens, particularly 17β-estradiol (E2), the most potent of the estrogens, may regulate cognitive function is through its ability to affect the number of parvalbumin (PV)-containing GABAergic interneurons. PV-interneurons are reduced in the brain of schizophrenia patients. In our previous study, ovariectomy (OVX) in mice reduced the number of hippocampal PV-interneurons which was accompanied by hippocampus-dependent memory impairment. Both neuron reduction and cognitive deficits were prevented by simultaneous E2 replacement after OVX. Due to the significant role of PV-interneurons in generating neuronal oscillations in the gamma frequency range, a vital component required for cognition, we investigated whether E2 can mediate gamma oscillations which would explain its influence on cognition. We further scrutinized whether raloxifene, a selective estrogen receptor modulator, has a similar effect on cognition as E2. Raloxifene has been shown to improve cognitive performance in schizophrenia patients and constitutes a safer treatment option as opposed to E2 due to its absence of peripheral side effects. We recorded electrical brain activities in the dorsal hippocampus of control, OVX mice or OVX mice with E2 or raloxifene implants both at baseline and during Y-maze, a hippocampal-dependent spatial memory task. While gamma-band oscillations were significantly increased in the control mice when placed in a novel environment (Y-maze), this increase was absent in OVX mice. E2 as well as raloxifene replacement prevented this deficit. This indicates that both E2 and raloxifene can regulate gamma oscillations in the dorsal hippocampus during exploration of a novel space. Moreover, OVX mice showed a significant reduction in gamma oscillations, specifically during decision making, which was accompanied by a significant deficit in short-term memory. E2 and raloxifene replacement rescued these deficits. This suggests that both E2 and raloxifene regulate spatial memory via specifically regulating hippocampal gamma oscillations during decision making. In agreement with this data we further demonstrated that raloxifene was able to recover gamma oscillations during decision making in the Polyinositic:polycitidylic acid (poly(I:C))-induced maternal immune activation mouse model of schizophrenia. Overall, these results suggest that raloxifene modulates hippocampus-dependent memory via preserving gamma oscillations through its conservation of PV-interneurons; a mechanism that most likely explains the beneficial effect of raloxifene on cognitive performance in schizophrenia patients. This aids understanding the mechanisms underlying the cognitive aspect of schizophrenia, but more importantly, strengthens the case of raloxifene as an adjunctive therapeutic option in this disorder.
Cell based restoration of motor function in rodent models of Parkinson's disease and stroke
Parkinson`s disease (PD) and Ischemic stroke are common disorders in our aging population. Although a large body of basic research highlights the potential of cell replacement therapy (CRT) as a novel therapeutic approach, clinical translation remains a distant goal. CRT has been most successfully applied in the (PD) field where clinical trials with human foetal tissue have established proof-of principle that new dopamine neurons implanted directly into the forebrain can provide significant relief from motor symptoms in some patients. Although the approach has been far from universally successful at the clinical level, more than 30 years of basic and clinical research in this area has led to a detailed understanding of the in vivo properties of foetal tissue grafts and key principles underlying successful therapeutic impact. The work in this thesis focusses predominantly on promoting brain repair through cell transplantation. A major component of my PhD involved developing and extensively characterizing the phenotype of a focal model of ischemia as well as assessing functional and structural repair of the brain after transplantation. This work involved extensive behavioral testing during 9 months of graft integration and detailed histological examination of the graft and host tissue. In an additional project, I have examined the immunological response that occurs when transplanting neural progenitors within and across strains of mice, as well as into immune-compromised animals – findings that hold important implications for the design and interpretation of pre-clinical cell transplantation studies. Finally, I conducted a study addressing the role of meningeal cells in the development of dopamine neurons in the midbrain, demonstrating that these cells secrete chemokines that influence differentiation and axon guidance – knowledge that I subsequently demonstrated could impact on the integration of transplanted neurons in an animal model of PD.
Investigating the effect of spatial separation on the detection of sounds in competition, by examining electrophysiological responses from the brainstem and auditory cortex
Human communication frequently takes place in noisy environments. In these environments, successful understanding of speech is dependent on an individual’s ability to extract and use spatial cues for separating speech from distracting noise. When speech and noise are separated spatially, the speech reception threshold (SRT) is reduced, and this is referred to in the literature as spatial release from masking (SRM). SRM is in most part due to acoustic cues arising from the differences in time and intensity of signals arriving at each of our two ears (i.e. interaural time (ITD) and interaural level (ILD) differences). ITDs and ILDs have in general been investigated through psychoacoustic studies. However, the electrophysiological correlates of these acoustic cues have only been investigated individually. For this reason, a novel experiment was designed to investigate the effect of spatial separation on the detection of target sound in competition with distractor stimuli in a more realistic experimental environment in which both ITD and ILD cues were present. The primary aim of this thesis was to determine whether it is possible to identify a neural representation of SRM in the electrophysiological responses recorded from either the brainstem or auditory cortex, or both, using experimental stimuli conveying ILD and ITD cues. This research was conducted in two primary studies. The first study investigated whether the frequency-following response (FFR) in response to the fundamental frequency (F0) of a speech sound could be used to demonstrate SRM at different signal-to-noise ratios (SNRs), and what role of attentional mechanisms might play in spatial processing. FFRs were recorded in eighteen normally hearing participants. Participants were presented through headphones with a synthesized steady-state vowel /u/ with an F0 of 110 Hz and a 250 ms duration at 60 dB SPL. This vowel was labelled as the target stimulus. To be able to measure the effects of attention, a deviant stimulus was interspersed randomly throughout the target stimuli. It was presented 5% of the time, at 52 dB SPL. The role of attention in SRM was measured in two phases. In the “attended” phase, participants were asked to count the number of deviants that occurred. It was assumed that, while identifying the number of deviant stimuli, the participant was actively listening to the stimuli. In the “non-attended” phase, participants were asked to ignore both the target and deviant stimuli, and any distractors. The distractors were two continuous different stories spoken by the same speaker. Target, deviant and distractor stimuli were convolved with head-related transfer functions (HRTFs) to create two spatial conditions: the co-located condition with targets, deviants and distractors coming from 0⁰ azimuth; and the separated condition with the targets and deviants at 0⁰, but with each distractor shifted to each side (± 90⁰ azimuth). Three SNRs were considered (-5, -0, and 5 dB). The amplitudes of the FFR in response to F0 were determined and analysed. The results of study 1 revealed a significant effect of spatial separation. The effect of spatial separation was found only at the lower SNR. Spatially separating maskers from the target stimuli resulted in a significant larger amplitude of the FFR in response to the target F0. The spatial advantage obtained objectively was equivalent to an SNR increase of 3.3 dB. A significant effect for attention was found when participants actively focused on the target, as demonstrated by larger FFR amplitudes. However, no significant interactions were found between spatial separation and the level of attention. The findings of the first study suggest that binaural processing relevant to SRM may be reflected by phase locked neural activity in the brainstem. However, this objective measure may only be noticeable in relatively noisy environments. Furthermore, SRM may start early in the central auditory pathways regardless of one attending to the target stimuli or not. This last observation means that - although this thesis focuses on adults - an extrapolation potentially could be made towards the use with younger individuals, however with consideration of their brain differences with adults and the AEPs evoked from those brains. The lack of dependence on attention might be beneficial in investigating SRM in this population, where it is difficult to keep attention and one has to rely on objective techniques that do not require attending to the target stimulus. Conversely, the lack of interaction with attention may mean that the mechanism responsible for the objective results may be different from the mechanism primarily responsible for SRM. To identify whether objective markers of SRM can be recorded in either the brainstem or cortex (or both), a second study was conducted. In the second study, auditory brainstem responses (ABRs), FFRs, and cortical auditory evoked potentials (CAEPs) were recorded simultaneously from thirteen normally hearing adults in response to 200 target stimulus blocks. Each target stimulus block comprised of a series of 11 tone complexes (TCs), with each TC having a specific F0 and a duration of 30 ms, separated by a 30 ms interstimulus interval (ISI), resulting in a target stimulus block with a total duration of 630 ms. The blocks were repeated every 1200 ms. Two different target stimulus block paradigms were considered; flat and staircase. The ‘flat’ blocks had TCs with a constant fundamental frequency F0 of 325 Hz (and harmonics up to 6 kHz). In the ‘staircase’ blocks, the fundamental frequency of each TCs was reduced in steps of 30 Hz from 475 to 175 Hz, again with harmonics up to 6 kHz. ABRs were recorded to the onsets of the 30-ms TCs. FFRs were recorded in response to the F0s of the TCs, and CAEPs to the onsets of the target stimulus blocks. The distractor blocks, in contrast, comprised of blocks of TCs that were similar in number and duration to the target stimuli, but randomized in their F0 distribution from 100 to 550 Hz and jittered in time (+/- 15 ms) around the onset of the target TC. Both target and distractor stimuli were convolved with head-related transfer functions (HRTFs) and presented under headphones. The target stimuli were presented at 0⁰ azimuth. The distractors were co-located (at 0⁰ azimuth) and spatially separated (at ±90⁰ azimuth) from the targets. The targets were presented at SNRs of -5, 0, 5, 10 and 15 dB SNR, and at 60 dB SPL. After extraction and analysis of ABR amplitudes and latencies, and FFR amplitudes, the results of the second study revealed a significant effect of SRM as seen in a decrease in ABR latency for both flat and staircase target stimuli when spatially separating maskers from the target. FFR amplitude (only measured with the flat stimuli) was significantly larger in the separated condition, and a significant decrease in CAEP latencies (for the staircase stimuli) was found, but only at the lowest tested SNR of -5 dB. These results, particularly the FFR, confirmed the results obtained in the first study, i.e. separating distractors from the target, regardless of the type of stimulus being used, resulted in enhancing FFR F0 amplitude. However, due to noisy data, the observations at the cortical level need to be confirmed in a follow-up study. The spatial advantage was equivalent to a SNR increase of 4.3 dB for FFR amplitude (for the flat stimuli), and 13.8 dB for ABR latency, 11.2 dB for CAEP P1 and 19.9 dB for CAEP N1 latencies (for the staircase stimuli). The findings of the second study suggest that it is possible objectively to record SRM in both the brainstem and auditory cortex simultaneously at lower SNRs. This suggests that the central auditory system is able to squelch background noise via processing of spatial information, and that this capacity is higher in more challenging listening environments. Taken together, the results from the first and second studies suggest that it is feasible to use electrophysiological measures as a means of investigating the central auditory mechanisms, which contribute to SRM in the brainstem and cortex simultaneously. It is speculated that SRM occurs mainly at the level of the brainstem and is present at -5 dB SNR (i.e. difficult listening environments). The finding that SRM was primarily at lower SNRs is in reality not a clinical concern, as lower SNRs represent the environments in which SRM is generally found to be beneficial for the listener. Potential applications may be found in developing an objective detection test for spatial processing disorder (SPD), a condition in which normal-hearing individuals are unable to exploit the binaural mechanism of SRM when listening in noisy environments, i.e., a deficiency in selectively attending to target sounds, which are not spatially co-located with distractor sounds. Further studies are needed to investigate the effects of attention and SRM on brainstem and cortical responses in different populations including children, elderly, and people with SPD.
An anatomical and single-cell gene expression characterisation of putative neurogenesis from nestin-expressing cells in the adult mouse midbrain
Due to significant midbrain dopamine (DA) cell loss in Parkinson’s disease (PD), it is generally believed the most effective and long-term treatment for PD motor symptoms will be midbrain DA cell replacement therapy, either by endogenous repair or cell transplantation. However, cell transplantation is hindered by failure of acquisition and maintenance of the DA phenotype by cells transplanted into the adult midbrain, and endogenous repair has not advanced very far because there appears to be very little or no neurogenesis and DA neurogenesis here. Evidence suggests Nestin-expressing neural precursor cells (NPCs) may give rise to neurones, including DA neurones in the adult midbrain, however this needs to be confirmed and underlying mechanisms established. The aims of my study were to: (1) Determine whether there is baseline neurogenesis and DA neurogenesis from Nestin+ cells in the adult mouse midbrain. (2) Investigate the gene expression profile of Nestin+ cells during different stages of putative ontogenesis in the adult mouse midbrain. (3) Test the possibility that putative neurogenesis and DA neurogenesis from Nestin+ cells in the adult mouse midbrain can be regulated by exogenous factors. To achieve these aims Nestin-expressing cells in the adult mouse midbrain were permanently labelled with enhanced yellow fluorescent protein (eYFP) by administering Tamoxifen to adult transgenic Nestin-CreERT2 x R26eYFP mice. Four-days to 8-months later eYFP+ cells were studied using a combination of whole-cell electrophysiology, single-cell qPCR and immunohistochemistry. eYFP+ cells expressed a range of genes consistent with birth, migration, neuronal and DA neuronal differentiation. Parsing the gene expression profiles of eYFP+ cells by their location in the midbrain indicated they arise anywhere throughout the midbrain and differentiate and integrate locally, rather than migrate long distances to populate midbrain nuclei. Most eYFP+ cells expressed mature neuronal genes, which was consistent also with their neuronal electrophysiology comprising action potentials and spontaneous post-synaptic currents. In comparison to neighbouring control (eYFP-) cells, eYFP+ cells expressed more immature neuronal genes (Pax6, Ngn2 & Msx1), which was also consistent with their more immature electrophysiology comprising hyper-excitability, hyperpolarized resting membrane potential, shorter duration sPSCs, and smaller membrane capacitance. Many (53%) eYFP+ cells expressed a combination of mature and immature neuronal genes at all time-points following Tamoxifen (an approximation of their age), including very ‘young’ cells (<2 months). Many (42%) of these young eYFP+ cells also had mature neuronal morphology (large cells with prominent processes) and electrophysiology. Nestin and Ki67 (a marker of cell division) were only expressed by large cells, and early on after Tamoxifen. Additionally, eYFP+ cells increased in number and size with time following Tamoxifen, and they decreased in number following administration of the anti-mitotic drug, Cytarabine (Ara-C). These data indicate that eYFP+ cells are capable of cell division. The level of expression of mature neuronal genes in eYFP+ cells also increased over a time-course of 7 months. In these respects, eYFP+ cells appear to proliferate, grow and differentiate into neurones, albeit slowly. To achieve the third aim of the study, bioinformatics analyses of the genes I identified as uniquely characterising eYFP+ cells (i.e. the proneuronal genes Pax6, Msx1 and Ngn2) highlighted valproic acid (VPA) as a drug that might regulate putative neurogenesis and DA neurogenesis from Nestin+ cells in the adult mouse midbrain. Infusion of VPA directly into the midbrain of adult mice for 2 weeks had no effect on the number of eYFP+ cells but significantly reduced the number of Pax6+, Pax6+/NeuN+ and eYFP+/NeuN+ cells. However, it also significantly reduced the number of NeuN+ cells generally (eYFP+ and eYFP-), pointing to a general effect of VPA on NeuN expression rather than neurogenesis from Nestin+ cells. Thus neither VPA nor targeting Pax6 appears to regulate of the number of eYFP+ cells or their differentiation into neurones in the adult mouse midbrain. In summary, my findings add to evidence of a small but significant population of Nestin-expressing cells in the adult mouse midbrain. While the origin of these cells remains unclear, my data suggest that: (1) some eYFP+ cells in the adult midbrain do undergo neurogenesis, albeit slowly; (2) Nestin expression in the adult midbrain is not limited to NPCs and classical neurogenesis, but also occurs in mature neurons, perhaps in relation to dendritic remodelling, cell death, or some other form of neural plasticity. 3) Neither VPA nor targeting Pax6 appears to regulate of the number of eYFP+ cells or their differentiation into neurones in the adult mouse midbrain. Further study of these cells may provide crucial information to assist DA cell replacement therapy for PD.
Sensorimotor changes in knee osteoarthritis: from muscle spindle function to brain organisation and activity
Knee osteoarthritis (OA) is a major cause of disability and it is a growing problem due to the aging population and rising obesity rates. Better understanding of the disease process is required to inform the most efficient and effective methods of treatment for knee OA. Knee OA is associated with changes in motor and sensory function including impaired proprioception and motor output. The underlying causes of these impairments in knee OA remain unclear. Little previous research has investigated whether the proprioceptive impairments associated with knee OA are localised to the knee joint or are generalised across multiple joints in both the upper and lower limb joints. Muscle spindle function and the central processing of muscle spindle related afferent input in the brain are integral to optimal proprioception, however muscle spindle function in people with knee OA has not previously been investigated. Many of the documented motor and sensory function changes associated with knee OA alter input from the periphery. These changes along with the functional or structural reorganisation of the brain associated with other conditions that affect control of movement indicate likely reorganisation of at least the motor cortex and possibly other brain regions. No previous study has investigated reorganisation of the motor cortex or changes in activation across the whole brain associated with knee OA. To investigate whether joint repositioning proprioceptive deficits are localised to the diseased joint or generalised across other joints in people with knee OA, 30 individuals with right knee OA and 30 healthy asymptomatic controls performed active joint repositioning tests of the knee, ankle and elbow. Participants with knee OA had a larger relative error for joint repositioning of the knee than the controls. Relative error did not differ between groups for the ankle or elbow. These results are consistent with a mechanism for proprioceptive change that is localised to the knee joint. Given the important contribution muscle spindles make to proprioception differences, the function of quadriceps, triceps surae and tibialis anterior muscle spindles between people with and without knee OA were investigated. Thirty individuals with knee OA and 30 healthy asymptomatic controls stood comfortably and blindfolded on a force plate, with mechanical vibration applied over the quadriceps, triceps surae or tibialis anterior muscles. Anterior-posterior displacement of centre of pressure was analysed. Although there were no differences between groups for trials with vibration applied to the quadriceps or tibialis anterior, participants with knee OA were initially perturbed more by triceps surae vibration and accommodated less to repeated exposure than controls. This indicates that people with knee OA have less potential to detect or compensate for disturbed input to triceps surae, possibly due to an inability to compensate using muscles spindles in the quadriceps muscle. Differences in the organisation of the motor cortex between people with and without knee OA and possible associations between cortical organisation and accuracy of performance of a motor task were investigated. Functional magnetic resonance imaging (fMRI) data were collected while 11 participants with right knee OA and seven asymptomatic controls performed three force-matching motor tasks involving: 1) quadriceps, 2) tibialis anterior, and 3) finger/thumb flexor muscles. fMRI data were used to map the loci of peak activation in the motor cortex during the three tasks and to assess whether there were differences in the organisation of the motor cortex between the groups for the three motor tasks. Task accuracy was also quantified. A more anterior representation of the knee, and an opposite relative position of the knee and ankle representations in the motor cortex were found in people with knee OA. Poorer performance of the knee task was associated with more anterior placement of motor cortex loci in both groups. To investigate differences in activation of brain regions involved in sensorimotor processing between people with and without knee OA, brain activation during force matching tasks of the knee, ankle, or hand was assessed. fMRI data were collected with the participants, protocol and methods described above for investigations of motor cortex organisation. Areas of activation that differed between groups for each of the three motor tasks across the whole brain were identified. Task accuracy was also quantified. The combination of findings of changes in brain activation largely localised to the knee and of similar levels of performance accuracy across the knee, ankle and hand in the OA group indicate that a combination of factors, including those specific to sensorimotor control, may underlie changes in brain activation during knee, and to a lesser extent, ankle movements in people with knee OA. Brain activation was either not different or minimally different during the hand and ankle tasks, respectively. This indicates differences in sensorimotor processing are largely specific to the knee and not a generalised phenomenon. Overall this thesis provides evidence that in knee OA: 1) proprioceptive impairments are localised to the knee joint and not generalised across the ankle and elbow joints, 2) there are changes to muscle spindle function associated with knee OA, 3) organisation of the motor cortex differs between people with and without knee OA and differing organisation of the motor cortex is related to motor task performance, and 4) there are differences in brain activation between people with and without knee OA across a broad sensorimotor network associated with knee movement and a small number of areas of differing brain activation associated with ankle movement.
Transcriptional control of myelin maintenance in the adult CNS
Oligodendrocyte differentiation and the myelination of axons are crucial aspects of vertebrate central nervous system development and function. The processes of oligodendrocyte differentiation and the expression of these myelin proteins are tightly controlled by a number of transcription factors. Although the transcription factors required for the generation of oligodendrocytes and CNS myelination during development have been relatively well established, it is not known whether continued expression of the same factors is required for the maintenance of myelin in the adult. Myelin Regulatory Factor (MyRF), a putative transcription factor, is critical for the generation of mature oligodendrocytes and the initiation of myelination during development. It has however not been investigated whether MyRF is also required for the ongoing maintenance of myelin once oligodendrocyte differentiation is complete. Moreover, whether it is really a transcription factor has also been unclear, given it contains a putative transmembrane domain and the ability of the MyRF protein to interact with DNA has not been demonstrated. The aims of this thesis were therefore to investigate the roles of MyRF in the mature CNS, and to determine the molecular mechanisms by which it promotes myelination. To investigate whether ongoing expression of MyRF is required to maintain a mature myelinated central nervous system, an inducible conditional knockout strategy was used to ablate MyRF specifically in mature oligodendrocytes of adult mice. This approach resulted in a rapid down-regulation of key myelin genes, followed by an eventual death of many of the recombined oligodendrocytes and a delayed, but severe, CNS demyelination. As such, MyRF has clear roles in regulating myelination beyond the initial phase of oligodendrocyte differentiation. At the molecular level, it was found that MyRF is a novel example of a membrane-tethered transcription factor which undergoes cleavage via a unique autoproteolytic mechanism previously undescribed in eukaryotic cells. This mechanism relies on a protein domain previously only reported in bacteriophage tailspike proteins. This cleavage of MyRF allows the N-terminal cleavage product to translocate to the nucleus, where it directly binds DNA via evolutionary conserved residues and directly promotes expression of myelin genes. Together, these data provide crucial insights into the biological roles of MyRF, establishing it as a novel type of transcription factor with a direct role in regulating the expression of genes required for myelination in both the developing and mature CNS.
Molecular mechanisms of relaxin in renal fibrosis
Renal fibrosis (scarring) is a hallmark of chronic kidney disease and is characterized by an excessive accumulation of extracellular matrix (ECM) proteins, primarily collagen. Driven by the continuous exposure to pathological stimuli and/or pro-fibrotic cytokines, fibrosis is a consequence of an imbalance between myofibroblast-induced ECM synthesis (fibrogenesis) and matrix metalloproteinase (MMP)-induced degradation (fibrolysis). Failure to resolve the progression of fibrosis causes significant organ dysfunction and the onset of failure. Angiotensin (Ang) II and transforming growth factor (TGF)-β1 are amongst the most potent cytokines that drive this pathological condition. To date, there are no effective treatments to prevent the ensuing structural and functional changes caused by fibrosis, with dialysis or transplantation as the only option for patients with end-stage kidney disease. The pregnancy-related hormone, relaxin, is now increasingly recognized as a rapid-occurring but safe anti-fibrotic agent. Relaxin has been consistently shown to inhibit Ang II- or TGF-β1-stimulated renal collagen deposition from numerous human and rodent fibroblast culture models in vitro, and in several experimental models of renal fibrosis in vivo, regardless of etiology. The anti-fibrotic properties of relaxin were found to be primarily mediated through its cognate receptor, Relaxin Family Peptide Receptor 1 (RXFP1) and via inhibition of TGF-β1-stimulated myofibroblast differentiation and myofibroblast-induced aberrant matrix/collagen synthesis, while promoting the expression/activity of several MMPs that can mediate collagen degradation.
An investigation of the role of the sympathetic nervous system in the pathophysiology of kidney dysfunction in severe sepsis
Sepsis is a leading cause of hospital morbidity and mortality causing organ failure with the kidney being commonly affected. Unfortunately, the pathophysiology of septic acute kidney injury (AKI) is poorly understood. Renal hypoperfusion has been traditionally proposed as the major pathophysiological mechanism of injury, but both human and animal research questions this belief. It is therefore critical to investigate alternative pathophysiological mechanisms causing septic AKI. Increasing evidence suggests that prolonged increased activity of the sympathetic nervous system (SNS) is detrimental to kidney function. Studies in rodent models of sepsis suggest that selective β1-adrenoceptor blockade improves mortality and centrally acting α2-adrenoceptor agonist therapy has recently been proposed for the treatment of septic AKI. The potential beneficial effect of these therapies may be due to inhibition of the action of the SNS on organ function or on the immune response, which can affect organ function via release of inflammatory cytokines. Currently little is known about the effects of excessive SNA in sepsis on the regulation of organ function. Accordingly, in my Ph.D. I aimed to assess the role of the SNS in septic organ failure, with particular focus on the kidney and heart. I addressed 4 questions: 1) What is the role of the SNS in determining the changes in renal function observed in sepsis? 2) Can selective β1-blockade be administered in sepsis without major haemodynamic consequences? 3) Can clonidine, a centrally acting α2-adrenoceptor agonist that decreases SNS outflow, restore renal function when administered in established sepsis? 4) What is the effect of sepsis on renal regional perfusion and oxygenation? To address these questions, I used an established ovine model of severe sepsis with a hypotensive, hyperdynamic state and AKI. Sepsis was induced with live Escherichia coli. Hyperdynamic sepsis was defined as tachycardia, vasodilatation with hypotension, and increases in cardiac output, arterial lactate level, temperature and respiratory rate. These alterations are similar to those observed in humans and they were achieved reproducibly. These changes were associated with reduced creatinine clearance (CreatCl) and urine output, despite increased total renal blood flow (RBF). Taken together, my studies showed no evidence that changes in renal SNS activation (RSNA) in sepsis contributed to the alterations in RBF or CreatCl. The studies indicate that the initial sepsis-induced diuresis resulted from inhibition of RSNA, but the final degree of oliguria was independent of the later increase in RSNA. Selective β1-adrenoceptor blockade did not adversely affect tissue perfusion, despite a significant reduction in heart rate, cardiac output and blood pressure, nor did it adversely affect RBF or renal function. Alpha2-agonism in established sepsis was safe. It transiently increased urine output in established sepsis without affecting CreatCl, thus suggesting a predominantly tubular effect. These findings suggest that this treatment could be safely tested in human trials. Finally, sepsis caused regional renal mismatches in perfusion and oxygenation in the cortex and medulla, with decreased perfusion and oxygenation observed in the latter, suggesting that septic AKI is a disease of the microcirculation rather than the macrocirculation.
Targeted ablation of Schwann cells: new insights into neuro-glial interactions
Schwann cells are specialised glial cells of the peripheral nervous system responsible for producing the myelin that ensheaths axons enabling salutatory conduction and providing neuroprotective and trophic support. In recent years, significant progress has been made into understanding the function of myelin and Schwann cells especially during development and in health, but several important questions concerning the mechanisms by which the peripheral nervous system responds to specific insults upon this myelinating cell population during adulthood remain unanswered. Currently, most experimental approaches to assess the consequences of myelinating Schwann cell death during adulthood that can be applied to answer some of these questions, use mechanisms that are non-specific, poorly-understood, result in animal death, and are often complicated by immune cell involvement or developmental onset. Such caveats make it difficult to understand the intricate sequence of degenerative and regenerative events and precise glial/axonal interactions responsible for functional outcomes in adult disease contexts. To address these technical limitations, a series of transgenic mouse lines were generated in the Merson laboratory to enable the specific inducible ablation of myelinating Schwann cells and oligodendrocytes, myelinating glial cells of the peripheral and central nervous systems, respectively. MBP-DTR transgenic mice express the human diphtheria toxin receptor (DTR) under the control of the proximal promoter of the murine myelin basic protein (MBP) gene, which is expressed in myelinating Schwann cells and oligodendrocytes. Challenging MBP-DTR mice with diphtheria toxin (DT) results in the specific ablation of myelinating glia via DT-mediated induction of apoptosis in DTR-expressing cells. Using this approach we have already demonstrated in transgenic line MBP-DTR100A, that oligodendrocytes undergo apoptosis resulting in rapid progressive terminal functional deficits within weeks of one intraperitoneal (i.p.) DT injection (10µg/kg). Histological analysis revealed that clinical decline was associated with a reduced density of oligodendrocyte cell bodies, alterations in the molecular and structural composition of nodes of Ranvier but no evidence of overt demyelination. There was some moderate signs of pathology in the peripheral nervous system as well, but this was not thoroughly characterised (Oluich et al., 2012). The major objective of the present work was to characterise both degenerative and regenerative responses to myelinating Schwann cell ablation. To enable such analyses, I have optimised several conditions across several MBP-DTR lines to identify conditions that elicit high survival rates that are appropriate for assessing both the early and late consequences of targeted Schwann cell death. Firstly, this thesis presents results demonstrating that I can modulate clinical parameters across MBP-DTR mouse lines dependent on DTR expression profile, DT dose and sex. From these experiments I have identified one line and one DT dosing paradigm of interest, the MBP-DTR25 line given 10µg/kg DT (i.p.) that elicits moderate non-lethal functional deficits, which is followed by rapid clinical recovery. MBP-DTR25+DT mice develop hindlimb weakness and a reduction in coordination, which peaks within 22-25 days and recovers rapidly by 28 days post-DT. To our surprise, we found that by clinical peak in MBP-DTR25+DT mice, myelinating Schwann cells had undergone apoptosis in the peripheral nervous system, but there was no signs of oligodendrocyte apoptosis in the central nervous system—nor apoptosis in wild-type+DT or MBPDTR25+saline control mice in either nervous systems, as expected. Such findings, illustrate tremendous regional specificity of this line and DT dosing paradigm— an aspect of the system that has been exploited to assess the precise consequences of specifically targeting myelinating Schwann cells in the peripheral nervous system. I have shown that at clinical peak there is; a reduction in the density of mature Schwann cell bodies, an increase in the density of demyelinated axons, evidence of myelin membrane swelling, an increase in the density of macrophages, but no difference in neuron or axon density. In addition, I have shown that mitochondria, sodium channels and potassium channels become redistributed and/or lost along the length of demyelinated axons. To assess whether remyelination plays a role in recovery, I have assessed the extent of Schwann precursor cell proliferation and remyelination. In the peripheral nervous system at 21, 28 and 35 days post-DT, MBP-DTR25+DT mice exhibited an increase in proliferative cells compared to wild-type+DT mice. At these time-points, a subset of proliferative cells were immunoreactive for Sox-10, indicating Schwann precursor cells enter the cell cycle after DT induced ablation of mature Schwann cells. Electron microscopy revealed evidence of remyelination at 28 and 35 days post-DT suggesting that responsive Schwann precursor cells mature rapidly into myelinating Schwann cells by acute clinical recovery. In conclusion, I have shown the precise dynamics of structural, functional and molecular responses of axons and the surrounding environment to targeted myelinating Schwann cell apoptosis at several timepoints post Schwann cell death. For the first time this has been shown in a model where non-specific direct damage to the axon does not confound outcomes, making this model ideal for understanding the precise cause-and-effect relationship between the axon and myelinating Schwann cells in the adult peripheral nervous system.