Magnetoencephalography:technical improvements in image co-registration and studies of visual cortical oscillations

The work presented in this thesis is divided into two distinct sections. In the first, the functional neuroimaging technique of Magnetoencephalography (MEG) is described and a new technique is introduced for accurate combination of MEG and MRI co-ordinate systems. In the second part of this thesis, MEG and the analysis technique of SAM are used to investigate responses of the visual system in the context of functional specialisation within the visual cortex. In chapter one, the sources of MEG signals are described, followed by a brief description of the necessary instrumentation for accurate MEG recordings. This chapter is concluded by introducing the forward and inverse problems of MEG, techniques to solve the inverse problem, and a comparison of MEG with other neuroimaging techniques. Chapter two provides an important contribution to the field of research with MEG. Firstly, it is described how MEG and MRI co-ordinate systems are combined for localisation and visualisation of activated brain regions. A previously used co-registration methods is then described, and a new technique is introduced. In a series of experiments, it is demonstrated that using fixed fiducial points provides a considerable improvement in the accuracy and reliability of co-registration. Chapter three introduces the visual system starting from the retina and ending with the higher visual rates. The functions of the magnocellular and the parvocellular pathways are described and it is shown how the parallel visual pathways remain segregated throughout the visual system. The structural and functional organisation of the visual cortex is then described. Chapter four presents strong evidence in favour of the link between conscious experience and synchronised brain activity. The spatiotemporal responses of the visual cortex are measured in response to specific gratings. It is shown that stimuli that induce visual discomfort and visual illusions share their physical properties with those that induce highly synchronised gamma frequency oscillations in the primary visual cortex. Finally chapter five is concerned with localization of colour in the visual cortex. In this first ever use of Synthetic Aperture Magnetometry to investigate colour processing in the visual cortex, it is shown that in response to isoluminant chromatic gratings, the highest magnitude of cortical activity arise from area V2.

A psychological investigation into the relationship between hemispheric functioning and specific written language difficulties (dyslexia), using pharmaceutical intervention techniques

This thesis attempts a psychological investigation of hemispheric functioning in developmental dyslexia. Previous work using neuropsychological methods with developmental dyslexics is reviewed ,and original work is presented both of a conventional psychometric nature and also utilising a new means of intervention. At the inception of inquiry into dyslexia, comparisons were drawn between developmental dyslexia and acquired alexia, promoting a model of brain damage as the common cause. Subsequent investigators found developmental dyslexics to be neurologically intact, and so an alternative hypothesis was offered, namely that language is abnormally localized (not in the left hemisphere). Research in the last decade, using the advanced techniques of modern neuropsychology, has indicated that developmental dyslexics are probably left hemisphere dominant for language. The development of a new type of pharmaceutical prep~ration (that appears to have a left hemisphere effect) offers an oppertunity to test the experimental hypothesis. This hypothesis propounds that most dyslexics are left hemisphere language dominant, but some of these language related operations are dysfunctioning. The methods utilised are those of psychological assessment of cognitive function, both in a traditional psychometric situation, and with a new form of intervention (Piracetam). The information resulting from intervention will be judged on its therapeutic validity and contribution to the understanding of hemispheric functioning in dyslexics. The experimental studies using conventional psychometric evaluation revealed a dyslexic profile of poor sequencing and name coding ability, with adequate spatial and verbal reasoning skills. Neuropsychological information would tend to suggest that this profile was indicative of adequate right hemsiphere abilities and deficits in some left hemsiphere abilities. When an intervention agent (Piracetam) was used with young adult dyslexics there were improvements in both the rate of acquisition and conservation of verbal learning. An experimental study with dyslexic children revealed that Piracetam appeared to improve reading, writing and sequencing, but did not influence spatial abilities. This would seem to concord with other recent findings, that deve~mental dyslexics may have left hemisphere language localisation, although some of these language related abilities are dysfunctioning.

Cellular delivery of hammerhead ribozymes

Hammerhead ribozymes are potent RNA molecules which have the potential to specifically inhibit gene expression by catalysing the trans-cleavage of mRNAs. However, they are unstable in biological fluids and cellular delivery poses a problem. Site-specific chemical modification of hammerhead ribozymes was evaluated as a means of enhancing biological stability. Chimeric, 2'-O-methylated ribozymes, containing only five unmodified ribonucleotides, were catalytically active in vitro (kcat = 1.46 min-1) and were significantly more stable in serum and lysosomal enzymes than unmodified (all-RNA) counterparts. Furthermore, they remained undegraded in cell-containing media for up to 8 hours. Stability enhancement allowed cellular uptake properties of radiolabelled ribozymes to be assessed following exogenous delivery. Studies in vulval and glial cell lines indicated that chimeric ribozymes became cell-associated via an inefficient process, which was energy and concentration dependant. A considerable proportion of ribozymes remained bound to cell-surface components, however, a small proportion (<1%) were internalised via mechanisms of adsorptive and / or receptor mediated endocytosis. Fluorescent microscopy indicated that ribozymes were localised within endosomal / lysosomal vesicles following cell entry. This was confirmed by immuno-electron microscopy, which allowed the detection of biotin-labelled ribozymes within the cell ultrastructure. Despite the predominant localisation within endocytic vesicles, a small proportion of internalised ribozymes appeared able to exit these compartments and penetrate target sites within the nucleus and cytoplasm. The ribozymes designed in this report were directed against the epidermal growth factor receptor mRNA, which is over-expressed in a malignant brain disease called glioblastoma multiforme. In order to examine the fate of ribozymes in the brain, the distribution of FITC-labelled ribozymes was examined following intra-cerebro ventricular injection to mice. FITC-ribozymes demonstrated high punctate pattern of distribution within the striatum and cortex, which appeared to represent localisation within cell bodies and dendritic processes. This suggested that delivery to glial cells in vivo may be possible. Finally, strategies were investigated to enhance the cellular delivery of ribozymes. Conjugation of ribozymes to anti~transferrin receptor antibodies improved cellular uptake 3-fold as a result of a specific interaction with transferrin receptors. Complexation with cationic liposomes also significantly improved cell association, however, some toxiclty was observed and this could be a limitation to their use. Overall, it would appear that hammerhead ribozymes can be chemically stabilised to allow direct exogenous administration in vivo. However, additional delivery strategies are probably required to improve cellular uptake, and thus, allow ribozymes to achieve their full potential as pharmaceutical agents. KEYWORDS: Catalytic

Oral delivery strategies for the controlled release of cimetidine

It is advantageous to develop controlled release dosage forms utilising site-specific delivery or gastric retention for those drugs with frequent or high dosing regimes. Cimetidine is a potent and selective H2 -reception antagonist used in the treatment of various gastrointestinal disorders and localisation in the upper gastrointestinal tract could significantly improve the drug absorption. Three strategies were undertaken to prepare controlled release systems for the delivery of cimetidine to the GI tract. Firstly, increasing the contact time of the dosage form with the mucus layer which coats the gastrointestinal tract, may lead to increased gastric residence times. Mucoadhesive microspheres, by forming a gel-like structure in contact with the mucus, should prolong the contact between the delivery system and the mucus layer, and should have the potential for releasing the drug in sustained and controlled manner. Gelatin microspheres were prepared, optimised and characterised for their physicochemical properties. Crosslinking concentration, particle size and cimetidine loading influenced drug release profiles. Particle size was influenced by surfactant concentration and stirring speed. Mucoadheisve polymers such as alginates, chitosans, carbopols and polycarbophil were incorporated into the microspheres using different strategies. The mucoadhesion of the microspheres was determined using in vitro surface adsorption and ex vivo rat intestine models. The surface-modification strategy resulted in highest levels of microsphere adhesion, with chitosan, carbopols and polycarbophil as the most successful candidates for improvement of adhesion, with over 70% of the microspheres retained ex vivo. Specific targeting agent UEA I lectin was conjugated to the surface of gelatin microspheres, which enhanced the adhesion of the microspheres. Alginate raft systems containing antacids have been used extensively in the treatment of gastro-oesophageal disease and protection of the oesophageal mucosa from acid reflux by forming a viscous raft layer on the surface of the stomach content, and could be an effective delivery system for controlled release of cimetidine.

Hearing protectors: a dilemma involving acoustics and personal safety

This thesis is concerned with the optimising of hearing protector selection. A computer model was used to estimate the reduction in noise exposure and risk of occupational deafness provided by the wearing of hearing protectors in industrial noise spectra. The model was used to show that low attenuation hearing protectors con provide greater protection than high attenuation protectors if the high attenuation protectors ore not worn for the total duration of noise exposure; or not used by a small proportion of the population. The model was also used to show that high attenuation protectors will not necessarily provide significantly greater reduction in risk than low attenuation protectors if the population has been exposed to the noise for many years prior to the provision of hearing protectors. The effects of earplugs and earmuffs on the localisation of sounds were studied to determine whether high attenuation earmuffs are likely to have greater potential than the lower attenuation earplugs for affecting personal safety. Laboratory studies and experiments at a foundry with normal-hearing office employees and noise-exposed foundrymen who had some experience of wearing hearing protectors showed that although earplugs reduced the ability of the wearer to determine the direction of warning sounds, earmuffs produced more total angular error and more confusions between left and right. !t is concluded from the research findings that the key to the selection of hearing protectors is to be found in the provision of hearing protectors that can be worn for a very high percentage of the exposure time by a high percentage of the exposed population with the minimum effect on the personal safety of the wearers - the attenuation provided by the protection should be adequate but not a maximum value.

Further studies of the visually evoked subcortical potential in man

The Visually Evoked Subcortical Potential, a far-field signal, was originally defined to flash stimulation as a triphasic positive-negative-positive complex with mean latencies of P21 N26.2 P33.6 (Harding and Rubinstein 1980). Inconsistent with its subcortical source however, the signal was found to be tightly localised to the mastoid. This thesis re-examines the earlier protocols using flash stimulation and with auditory masking establishes by topographic studies that the VESP has a widespread scalp distribution, consistent with a far-field source of the signal, and is not a volume-conducted electroretinogram (ERG). Furthermore, mastoid localisation indicates auditory contamination from the click, on discharge of the photostimulator. The use of flash stimulation could not precisely identify the origin of the response. Possible sources of the VESP are the lateral geniculate body (LGB) and the superior colliculus. The LGB received 80% of the nerve fibres from the retina, and responds to high contrast achromatic stimulation in the form of drifting gratings of high spatial frequencies. At low spatial frequencies, it is more sensitive to colour. The superior colliculus is insensitive to colour and suppressed by contrast and responds to transitory rapid movements, and receives about 20% of the optic nerve fibres. A pattern VESP was obtained to black and white checks as a P23.5 N29.2 P34 complex in 93% of normal subjects at an optimal check size of 12'. It was also present as a P23.0 N28.29 P32.23 complex to red and green luminance balanced checks at 2o check size in 73% of subjects. These results were not volume-conducted pattern electroretinogram responses. These findings are consistent with the spatial frequency properties of the lateral geniculate body which is the considered source of the signal. With further work, the VESP may supplement electrodiagnosis of post-chiasmal lesions.

Psychophysical investigations of human peripheral vision

This thesis investigates various aspects of peripheral vision, which is known not to be as acute as vision at the point of fixation. Differences between foveal and peripheral vision are generally thought to be of a quantitative rather than a qualitative nature. However, the rate of decline in sensitivity between foveal and peripheral vision is known to be task dependent and the mechanisms underlying the differences are not yet well understood. Several experiments described here have employed a psychophysical technique referred to as 'spatial scaling'. Thresholds are determined at several eccentricities for ranges of stimuli which are magnified versions of one another. Using this methodology a parameter called the E2 value is determined, which defines the eccentricity at which stimulus size must double in order to maintain performance equivalent to that at the fovea. Experiments of this type have evaluated the eccentricity dependencies of detection tasks (kinetic and static presentation of a differential light stimulus), resolution tasks (bar orientation discrimination in the presence of flanking stimuli, word recognition and reading performance), and relative localisation tasks (curvature detection and discrimination). Most tasks could be made equal across the visual field by appropriate magnification. E2 values are found to vary widely dependent on the task, and possible reasons for such variations are discussed. The dependence of positional acuity thresholds on stimulus eccentricity, separation and spatial scale parameters is also examined. The relevance of each factor in producing 'Weber's law' for position can be determined from the results.

Topographic studies of scalp potentials evoked by pattern presentation

The waveform and scalp distribution of the visual evoked potentials elicited by stimuli in the foveal and parafoveal regions have been investigated in a group of normal humans using a 16-channel `brain mapping' system. The waveform and topography of the responses to pattern onset and pattern reversal stimulation were investigated, using 4 x 4o full field and 4 x 2o lateral and altitudinal half-field stimuli. The responses were composed of several successive peaks which are in some respects consistent with those demonstrated by other workers using larger field sizes. The differences in the behaviour of these components with respect to the position of the stimulus in the visual field were suggestive of origins in different areas of the visual cortex and/or different visual mechanism. Of particular interest were the major early positive components `P90' and `P95' of the responses to pattern onset and pattern reversal stimulation respectively. More detailed exploration of the behaviour of these major early positive components was carried out using `M-scaled' stimuli selected to activate one square centimetre patches of striate cortex and associated extrastriate re-projections, positioned at different points in the foveal and parafoveal area of the visual field. The inter- and intra-subject variability in amplitude and localisation of the signals elicited by these targets was considered to be a reflection of the individual variations in relationship of visual field projections with the pattern of gyri and fissures on the proximal surface of the occipital lobe. The behaviour of component P90 of the onset response is consistent with a lateral origin in extrastriate visual cortex; that of P95 of the pattern reversal response is consistent in some respects with a striate cortical origin, but in others with a partial origin in extrastriate cortex.

Visual evoked magnetic responses (VEMR) to flash and pattern reversal stimulation

The problems of using a single channel magnetometer (BTi, Model 601) in an unshielded clinical environment to measure visual evoked magnetic responses (VEMR) were studied. VEMR to flash and pattern reversal stimuli were measured in 100 normal subjects. Two components, the P100M to pattern reversal and P2M to flash, were measured successfully in the majority of patients. The mean latencies of these components in different decades of life were more variable than the visual evoked potentials (VEP) that have been recorded to these stimuli. The latency of the P100M appeared to increase significantly after about 55 years of age whereas little change occurred for the flash P2M. The effects of blur, check size, stimulus size and luminance intensity on the latency and amplitude of the VEMR were studied. Blurring a small (32') check significantly increased latency whereas blurring a large (70') check had little effect on latency. Increasing check size significantly reduced latency of the P100M but had little effect on amplitude. Increasing the field size decreases the latency and increases the amplitude of the P100M. Within a normal subject, most of the temporal variability of the P100M appeared to be associated with run to run variation rather than between recording sessions on the same day or between days. Reproducibility of the P100M was improved to a degree by employing a magnetically shielded room. Increasing flash intensity decreases the latency and increases the amplitude of the P2M component. The magnitude of the effects of varying stimulus parameters on the VEMR were frequently greater than is normally seen in the VEP. The topography of the P100M and P2M varied over the scalp in normal subjects. Full field responses to a large check could be explained as approximately the sum of the half field responses and were consistent with the cruciform model of the visual cortex. Preliminary source localisation data suggested a shallower source in the visual cortex for the flash P2M compared with the P100M. The data suggest that suitable protocols could be devised to obtain normative data of sufficient quality to use the VEMR to flash and pattern clinically.

The role of TG2 in ECV304-related vasculogenic mimicry

Tumour vasculogenesis can occur by a process referred to as vasculogenic mimicry, whereby the vascular structures are derived from the tumour itself. These tumours are highly aggressive and do not respond well to anti-angiogenic therapy. Here, we use the well characterised ECV304 cell line, now known as the bladder cancer epithelial cell line T24/83 which shows both epithelial and endothelial characteristics, as a model of in vitro vasculogenic mimicry. Using optimised ratios of co-cultures of ECV304 and C378 human fibroblasts, tubular structures were identifiable after 8 days. The tubular structures showed high levels of TG2 antigen and TG in situ activity. Tubular structures and in situ activity could be blocked either by site-directed irreversible inhibitors of TG2 or by silencing the ECV304 TG2 by antisense transfection. In situ activity for TG2 showed co-localisation with both fibronectin and collagen IV. Deposition of these proteins into the extracellular matrix could be reduced by inclusion of non-cell penetrating TG inhibitors when analysed by Western blotting suggesting that the contribution of TG2 to tube formation is extracellular. Incubation of ECV304 cells with these same irreversible inhibitors reduced cell migration which paralleled a loss in focal adhesion assembly, actin cytoskeleton formation and fibronectin deposition. TG2 appears essential for ECV304 tube formation, thus representing a potential novel therapeutic target in the inhibition of vasculogenic mimicry. © 2012 Springer-Verlag.

Effective electromagnetic noise cancellation with beamformers and synthetic gradiometry in shielded and partly shielded environments

The major challenge of MEG, the inverse problem, is to estimate the very weak primary neuronal currents from the measurements of extracranial magnetic fields. The non-uniqueness of this inverse solution is compounded by the fact that MEG signals contain large environmental and physiological noise that further complicates the problem. In this paper, we evaluate the effectiveness of magnetic noise cancellation by synthetic gradiometers and the beamformer analysis method of synthetic aperture magnetometry (SAM) for source localisation in the presence of large stimulus-generated noise. We demonstrate that activation of primary somatosensory cortex can be accurately identified using SAM despite the presence of significant stimulus-related magnetic interference. This interference was generated by a contact heat evoked potential stimulator (CHEPS), recently developed for thermal pain research, but which to date has not been used in a MEG environment. We also show that in a reduced shielding environment the use of higher order synthetic gradiometry is sufficient to obtain signal-to-noise ratios (SNRs) that allow for accurate localisation of cortical sensory function.

Numerical investigation of the effect of the temporal pulse shape on modification of fused silica by femtosecond pulses

We report the results of numerical studies of the impact of asymmetric femtosecond pulses focused in the bulk of the material on the femtosecond modification of fused silica. It is shown that such pulses lead to localisation of absorption in the process of femtosecond modification and to a decrease in the threshold energy of modification. It is found that the optimal asymmetry parameters for reaching the maximum plasma density in the focusing region depend on the pulse energy: at an initial energy of about 100 nJ, it is preferable to use pulses with positive TOD; however, when the energy is increased, it is preferable to use pulses with negative TOD. This is explained by differences in the dynamics of the processes of absorption of energy of a pulse propagating in the material.

Lymphoid aggregates that resemble tertiary lymphoid organs define a specific pathological subset in metal-on-metal hip replacements

Aseptic lymphocyte-dominated vasculitis-associated lesion (ALVAL) has been used to describe the histological lesion associated with metal-on-metal (M-M) bearings. We tested the hypothesis that the lymphoid aggregates, associated with ALVAL lesions resemble tertiary lymphoid organs (TLOs). Histopathological changes were examined in the periprosthetic tissue of 62 M-M hip replacements requiring revision surgery, with particular emphasis on the characteristics and pattern of the lymphocytic infiltrate. Immunofluorescence and immunohistochemistry were used to study the classical features of TLOs in cases where large organized lymphoid follicles were present. Synchrotron X-ray fluorescence (XRF) measurements were undertaken to detect localisation of implant derived ions/particles within the samples. Based on type of lymphocytic infiltrates, three different categories were recognised; diffuse aggregates (51%), T cell aggregates (20%), and organised lymphoid aggregates (29%). Further investigation of tissues with organised lymphoid aggregates showed that these tissues recapitulate many of the features of TLOs with T cells and B cells organised into discrete areas, the presence of follicular dendritic cells, acquisition of high endothelial venule like phenotype by blood vessels, expression of lymphoid chemokines and the presence of plasma cells. Co-localisation of implant-derived metals with lymphoid aggregates was observed. These findings suggest that in addition to the well described general foreign body reaction mediated by macrophages and a T cell mediated type IV hypersensitivity response, an under-recognized immunological reaction to metal wear debris involving B cells and the formation of tertiary lymphoid organs occurs in a distinct subset of patients with M-M implants. © 2013 Mittal et al.

Cellular uptake of ribonuclease A-functionalised core-shell silica microspheres

Analysis of protein function in a cellular context ideally requires physiologically representative levels of that protein. Thus conventional nucleic acid-based transfection methods are far from ideal owing to the over expression that generally results. Likewise fusions with protein transduction domains can be problematic whilst delivery via liposomes/nanoparticles typically results in endosomal localisation. Recently polymer microspheres have been reported to be highly effective at delivering proteins into cells and thus provide a viable new alternative for protein delivery (protein transduction). Herein we describe the successful delivery of active ribonuclease A into HeLa cells via novel polymer core-silica shell microspheres. Specifically, poly(styrene-co-vinylbenzylisothiouronium chloride) core particles, generated by dispersion polymerisation, were coated with a poly(styrene-co-trimethoxysilylpropyl methacrylate) shell. The resultant core-shell morphology was characterised by transmission electron, scanning electron and fluorescence confocal microscopies, whilst size and surface charge was assessed by dynamic light scattering and zeta-potential measurements, respectively. Subsequently ribonuclease A was coupled to the microspheres using simple carbodiimide chemistry. Gel electrophoresis confirmed and quantified the activity of the immobilised enzyme against purified HeLa RNA. Finally, the polymer-protein particles were evaluated as protein-transduction vectors in vitro to deliver active ribonuclease A to HeLa cells. Cellular uptake of the microspheres was successful and resulted in reduced levels of both intracellular RNA and cell viability.