Axonal excitability in disorders of the peripheral and central nervous system

Peripheral axonal excitability techniques have given pathophysiological insights into many peripheral nerve disorders, with clinical application still in its infancy. Small excitability changes are usually seen compared to controls, raising questions of sensitivity. The remote effects of central lesions on peripheral excitability are not well defined. The purpose of this thesis was to correlate the changes of excitability in peripheral neuropathic disorders with their clinical signs and nerve conduction findings, and to examine for changes in peripheral nerves in a predominantly central nervous disease model. Three peripheral disorders were studied in this way for the first time. To look for a central effect on peripheral nerves, multiple sclerosis (MS) was studied, and compared with other lesional central nervous disorders. Ischaemic depolarisation was suggested in end-stage liver disease and this was not reversible one year after liver transplantation. Similar findings were noted in HIV-positive subjects but only in nucleoside drug-related neuropathy, distinguishing it from distal sensory polyneuropathy. In mitochondrial disease, motor studies showed no changes at rest or with experimental ischaemia. Sensitivity and subset comparisons indicate that in liver disease, some excitability changes correlated with peripheral clinical signs but not standard nerve conduction abnormalities. The reverse was true in mitochondrial disease. Roughly 20-25% of patients with end-stage liver neuropathy and nucleoside neuropathy were identified to fall outside control 95% confidence interval limits. Upregulated slow K+ channels seen in peripheral motor axons of MS are possibly a response to enhanced persistent inward currents (PICs) at the motoneuron following suprasegmental input interruption. In contrast, peripheral sensory studies show increased fast K+ conductance through altered gating kinetics, possibly because of humoral factors acting locally to loosen the paranodal seal

The Translational Studies of Pain: From Spinal Neurones to Human Perception

The discovery of new treatments for chronic pain relies on the detection of pre-clinical targets and the progression to successful clinical trials. In order to improve this transition reliable translational models must be identified, based on mechanisms that underlie the symptoms of chronic pain. This thesis aimed to validate the use of 3 potential translational models: topical capsaicin, ultraviolet irradiation (UVB) and UVB rekindling. Furthermore, using a mechanism based approach to treatment, the modulation of capsaicin induced sensitisation was explored in animals and humans. In order to characterise the models in rats, in vivo electrophysiological recordings were made from single unit dorsal horn wide dynamic range neurones. Evoked responses to thermal, mechanical and electrical stimulation were quantified. To complement the animal studies, full QST profiling was undertaken on healthy human volunteers. Assessments of the pain thresholds were made, as well as numerical ratings to sub and supra threshold stimuli, in order to best compare these results with rodent data. All of the models tested evoked similar sensory changes across species, and the symptoms induced in each of the models were used to infer the peripheral and central components. Sensory changes evoked by capsaicin included mechanical hypersensitivity accompanied by a facilitation of responses in the Aδ fibre range. These are reflective of both a peripheral and central sensitisation. Furthermore, these changes were prevented by pre-treatment with the adenosine receptor 1 (A1R) agonist, CPA. UVB appeared to be a strictly peripheral model, resulting in no secondary changes or receptive field expansion. On the other hand, the UVB rekindling model showed clear signs of engaging both peripheral and central mechanisms, including thermal allodynia, secondary brush hypersensitivity and a facilitation of Aβ fibre responses. Overall, we confirmed that similar short-term sensory consequences, that may mimic certain pathophysiologies, could be engaged and quantified in rats and human volunteers in response to topical capsaicin, UVB irradiation and UVB rekindling. The UVB rekindling model induced signs of the engagement of a number of clinically relevant phenomena, such as peripheral inflammation/ sensitisation driving central modifications. As such this model will be useful in investigating mechanisms of inflammatory pain and assessing analgesic efficacy of novel medications

Neuromuscular fatigue, muscle temperature and hypoxia: an integrative approach.

Real world exposures to physiologically and/or psychologically stressful environments are often multifactorial. For example, high-altitude typically combines exposure to hypobaric hypoxia, solar radiation and cold ambient temperatures, while sea level thermal stress is often combined with supplementary or transient stressors such as rain, solar radiation and wind. In such complex environments, the effect of one stressor on performance may be subject to change, simply due to the presence of another independent stressor. Such differential influences can occur in three basic forms; additive, antagonistic and synergistic, each term defining a fundamental concept of inter-parameter interactions. As well as the natural occurrence of stressors in combination, understanding interactions is fundamental to experimentally modelling how multiple physiological strains integrate in their influence on or regulation of - exercise intensity. In this thesis the current literature on neuromuscular fatigue and the influence of thermal and hypoxic stress is reviewed (Chapter 1). This is followed by an outline of the methodological developments used in the subsequent experiments (Chapter 2). In the first experimental study (Chapter 3) a novel approach was adopted to investigate the combined effect of muscle cooling and hypoxia on neuromuscular fatigue in humans. The results showed that the neuromuscular system s maximal force generating capacity declined by 8.1 and 13.9% during independent cold and hypoxic stress compared to control. Force generation decreased by 21.4% during combined hypoxic-cold compared to control, closely matching the additive value of hypoxia and cold individually (22%). This was also reflected in the measurement of mechanical fatigue (electromechanical ratio), demonstrating an additive response during combined hypoxic-cold. From this study, it was concluded that when moderate hypoxia and cold environmental temperatures are combined during low intensity exercise, the level of fatigue increases additively with no interaction between these stressors. Before conducting a more complex investigation on combined stressors, a better understanding of the role of muscle temperature on central fatigue - i.e. voluntary muscle activation via the afferent signalling pathways was sought. The focus of Chapter 4 was to quantify the relationship between muscle temperature and voluntary muscle activation (central fatigue) across a wide range of temperatures. The primary finding was that different muscle temperatures can induce significant changes in voluntary activation (0.5% reduction per-degree-centigrade increase in muscle temperature) when neural drive is sustained for a prolonged effort (e.g. 120-s); however this effect is not exhibited during efforts that are brief in duration (e.g. 3-s). To further explore this finding, Chapter 5 investigated the effect of metaboreceptive feedback at two different muscle temperatures, using post-exercise muscle ischemia, on voluntary activation of a remote muscle group. The results showed that at the same perceived mental effort, peripheral limb discomfort was significantly higher with increasing muscle temperature (2% increase per-degree-centigrade increase). However any influence of increased muscle temperature on leg muscle metaboreceptive feedback did not appear to inhibit voluntary muscle activation - i.e. central control - of a remote muscle group, as represented by an equal force output and voluntary activation in the thermoneutral, contralateral leg. In Chapter 6, the psycho-sensory effects of changes in muscle temperature on central fatigue during dynamic exercise were investigated. During sustained dynamic exercise, fatigue development appeared to occur at a faster rate in hot muscle (4% increase per-degree-centigrade increase) leading to a nullification of the beneficial effects of increased muscle temperature on peak power output after a period of ~60-s maximal exercise. In support of previous studies using isometric exercise (Chapter 4 and 6), participants reported significantly higher muscular pain and discomfort in hot muscle compared to cooler muscle during dynamic exercise (2 and 1% increase per-degree-centigrade increase respectively), however this did not result in a lower power output. From Chapters 4, 5 and 6 it was concluded that in addition to faster rates of metabolite accumulation due to cardiovascular strain, it is possible that a direct sensitisation of the metaboreceptive group III and IV muscle afferents occurs in warmer muscle. This likely contributes to the reduction in voluntary muscle activation during exercise in the heat, while it may attenuate central fatigue in the cold. It was also interpreted that muscle afferents may have a similar signalling role to cutaneous sensory afferents; the latter of which are recognised for their role in providing thermal feedback to the cognitive-behavioural centres of the brain and aiding exercise regulation under thermal stress. The impact of body core and active muscle temperature on voluntary muscle activation represented a similar ratio (5 to 1 respectively) to the temperature manipulated (single leg) to non-temperature manipulated mass (rest of body) in Chapters 4, 5 and 6. This indicates that voluntary muscle activation may also be regulated based on a central meta-representation of total body heat content i.e. the summed firing rates of all activated thermoreceptors in the brain, skin, muscle, viscera and spine. Building on the initial findings of Chapter 3, Chapter 7 investigated the causative factors behind the expression of different interaction types during exposure to multi-stressor environments. This was achieved by studying the interaction between thermal stress and hypoxia on the rate of peripheral and central fatigue development during a high intensity bout of knee extension exercise to exhaustion. The results showed that during combined exposure to moderate hypoxia and mild cold, the reductions in time to exhaustion were additive of the relative effects of hypoxia and cold independently. This differs from the findings in Chapter 3, in which fatigue was additive of the absolute effects of cold and hypoxia. In contrast, combining moderate hypoxia with severe heat stress resulted in a significant antagonistic interaction on both the absolute and relative reductions in time to exhaustion i.e. the combined effect being significantly less than the sum of the individual effects. Based on the results in Chapter 7, a quantitative paradigm for understanding of systematic integration of multifactorial stressors was proposed. This is, that the interaction type between stressors is influenced by the impact magnitude of the individual stressors effect on exercise capacity, whereby the greater the stressors impact, the greater the probability that one stressor will be cancelled out by the other. This is the first study to experimentally model the overarching principles characterising the presence of simultaneous physiological strains, suggesting multifactorial integration be subject to the worst strain takes precedence when the individual strains are severe

The function of NaV1.8 clusters in lipid rafts

NaV1.8 is a voltage gated sodium channel mainly expressed on the membrane of thin diameter c-fibre neurons involved in the transmission of pain signals. In these neurons NaV1.8 is essential for the propagation of action potentials. NaV1.8 is located in lipid rafts along the axons of sensory neurons and disruption of these lipid rafts leads to NaV1.8 dependant conduction failure. Using computational modelling, I show that the clustering of NaV1.8 channels in lipid rafts along the axon of thin diameter neurons is energetically advantageous and requires fewer channels to conduct action potentials. During an action potential NaV1.8 currents across the membrane in these thin axons are large enough to dramatically change the sodium ion concentration gradient and thereby void the assumptions upon which the cable equation is based. Using scanning electron microscopy NaV1.8 is seen to be clustered, as are lipid raft marker proteins, on neurites at scales below 200nm. FRET signals show that the lipid raft marker protein Flotillin is densely packed on the membrane however disruption of rafts does not reduce the FRET signal from dense protein packing. Using mass spectrometry I investigated the population of proteins found in the lipid rafts of sensory neurons. I found that the membrane pump NaK-ATPase, which restores the ion concentrations across the membrane, is also contained in lipid rafts. NaK-ATPase may help to offset concentration changes due to NaV1.8 currents enabling the repeated firing of c-fibres, which is associated with spontaneous pain in chronic pain disorders.Open Acces

Modelling changes in excitability of the peripheral nervous system using compartmentalised microfluidic culture

This thesis describes the use of compartmentalised microfluidic devices to investigate changes in neuronal excitability. All studies carried out in this work were completed in line with principles of the NC3Rs (reduction, replacement and refinement). Particular interest was given to the study of the excitability of dorsal root ganglion neurons (DRGs) in the context of pain-based signalling. This also included the in vitro culture and characterisation of non-neuronal cells involved in inflammation and nociception. Current methods for In vitro modelling of pain pathways often fails to replicate the unique morphology of the DRG neurons. These pseudo-unipolar neurons detect nociceptive stimuli at the peripheral terminals, and transduce long range action-potentials to higher processing centres in the central nervous system. Unlike in vivo modelling of pain behaviours, in vitro models of nociception provide the capacity to monitor changes in neuronal function at a cellular and molecular level. However, until the development of technology such as microfluidics, the standard methods of culture failed to isolate the axons from the soma. The primary aim of this project was to develop a model capable of replicating the complex microenvironment that terminals of the DRG neurons encounter during the development and onset of pain. This involved the optimisation of cell culture methods for inflammatory cells used to induce changes in neuronal excitability, both from the context of the peripheral terminals, or from the CNS if desired. At a molecular level, the microfluidic model was also used to investigate the role of small non-coding RNA (microRNAs) on regulating DRG excitability in the context of nociception. This Thesis hypothesises that voltage-gated potassium channels form an interesting target for a microRNA of interest. However, it is widely acknowledged that microRNAs regulate the expression of multiple mRNAs. The use of functional studies using the microfluidic model have shown here that there are differences in the way in which a neuron responds to a stimulus, dependent on whether it is applied locally to the axon or the soma. Live cell imaging was used to measure evoked changes in Ca2+ transients as a proxy for cell excitability. As well as significant differences in the response to depolarising agents such as potassium chloride (KCL), the use of biologically relevant stimuli to the study of nociception was also developed. The culture of inflammatory cells such as bone marrow derived macrophages led to the development of cytokine-rich media which was used to evoke changes in neuronal excitability. By exploiting the microfluidic nature of the device, subsequent investigations to the role of microRNA 138-5p in regulating neuronal excitability were undertaken. The use of cell permeable microRNA inhibition showed a reduction in cell excitability if applied locally to the axons. Bioinformatics led to the development of Kv1.2 as a potential target for miR-138-5p in vivo, which could explain the effects of miR-138-5p in modulating excitability of the DRGs. The findings in this work have demonstrated the potential for development of more biologically relevant in vitro models using microfluidic compartmentalised cell culture. For example, fluidic isolation has characterised the role of miR-138-5p in regulating DRG excitability at the axons

Neuro-glia and the delayed onset of pain-like hypersensitivity following infant nerve injury

Peripheral nerve injuries in adults can trigger neuropathic pain which coincides with alterations in dorsal horn neuronal activity and glial cells residing in the dorsal horn, and infiltrating T-lymphocytes. These cells synthesise and release pro-inflammatory mediators that can directly and indirectly sensitize dorsal horn neurones and contribute to neuropathic pain. Neuropathic pain is rare in infants and studies presented in this thesis show that a spared nerve injury (SNI) at postnatal day (P) 10 does not result in pain-like behaviour but triggers an anti-inflammatory immune response in the dorsal horn, not observed in nerve injured adults. However, if infant SNI treated animals are intrathecally administered with the pro-inflammatory mediator tumor necrosis factor (TNF) or lipopolysaccharide-activated microglia they develop pain-like behaviour while blockade of anti-inflammatory activity after infant SNI ‘unmasks’ neuropathic pain-like behaviour. Thus, nerve injury induced pain-like hypersensitivity in infants is actively suppressed by dominant anti-inflammatory neuro-immune activity. The anti-inflammatory response can also be evoked by direct C-fibre nerve stimulation in the infant, but not in adult rodents. However, mechanical hypersensitivity does eventually develop following early life nerve injury in the rat at adolescence (Vega-Avelaira et al., 2012). Longitudinal studies presented in this thesis indicate that hypersensitivity emerges in response to not only mechanical stimulation but also following innocuous and noxious cold stimulation of the hind paw, and contralateral weight bearing. The emergence of behavioural hypersensitivity at adolescence coincides with an increase in spontaneous and evoked- activity of wide dynamic dorsal horn neurons, that is absent in sham controls. In addition, the immune response in the dorsal horn switches from an anti-inflammatory response to pro-inflammatory, characterised by an increase in the expression of TNF and Brain-derived neurotrophic factor. This explains why neuropathic pain is rare in infants, but complex regional pain syndromes can emerge, for no observable reason at adolescence

Comparison of Multi-Compartment Cable Models of Human Auditory Nerve Fibers

Background: Multi-compartment cable models of auditory nerve fibers have been developed to assist in the improvement of cochlear implants. With the advancement of computational technology and the results obtained from in vivo and in vitro experiments, these models have evolved to incorporate a considerable degree of morphological and physiological details. They have also been combined with three-dimensional volume conduction models of the cochlea to simulate neural responses to electrical stimulation. However, no specific rules have been provided on choosing the appropriate cable model, and most models adopted in recent studies were chosen without a specific reason or by inheritance. Methods: Three of the most cited biophysical multi-compartment cable models of the human auditory nerve, i.e., Rattay et al. (2001b), Briaire and Frijns (2005), and Smit et al. (2010), were implemented in this study. Several properties of single fibers were compared among the three models, including threshold, conduction velocity, action potential shape, latency, refractory properties, as well as stochastic and temporal behaviors. Experimental results regarding these properties were also included as a reference for comparison. Results: For monophasic single-pulse stimulation, the ratio of anodic vs. cathodic thresholds in all models was within the experimental range despite a much larger ratio in the model by Briaire and Frijns. For biphasic pulse-train stimulation, thresholds as a function of both pulse rate and pulse duration differed between the models, but none matched the experimental observations even coarsely. Similarly, for all other properties including the conduction velocity, action potential shape, and latency, the models presented different outcomes and not all of them fell within the range observed in experiments. Conclusions: While all three models presented similar values in certain single fiber properties to those obtained in experiments, none matched all experimental observations satisfactorily. In particular, the adaptation and temporal integration behaviors were completely missing in all models. Further extensions and analyses are required to explain and simulate realistic auditory nerve fiber responses to electrical stimulation