Anatomy and Neuroscience - Theses
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ItemNo Preview AvailableTracing Diet and Mobility of Past Human Populations in Greater Mtskheta, Georgia( 2021)The region of Greater Mtskheta (Republic of Georgia) lies in the Southern Caucasus and presents a near continuous record of human occupation throughout the Late Bronze-Early Iron Age (LBA-EIA, 1500-500 BC), Hellenistic Period (400-1 BC) and Roman-Late Antique Period (RLA; AD 1-700). Greater Mtskheta became increasingly urbanised, densely populated and hosted an increasingly complex society during these time periods. Contemporary written sources provide little insight into the lifestyles and social organisation maintained by Mtskheta’s inhabitants; researchers rely heavily on the trace remains of Mtskheta settlements and cemeteries to reconstruct how the inhabitants lived and what resources they consumed. Archaeological investigations reveal that by the 1st century AD, the resident society was multi-cultural, socially stratified and maintained far-reaching trade networks with the Greeks, Romans, Parthians, and Sassanid Persians. Following the 4th century AD a series of cultural changes emerge in Greater Mtskheta cemeteries including a shift in burial customs, the appearance of people with intentionally modified crania and new ‘Eurasian’ styles of grave goods. These changes suggest a novel cultural influence arrived in Mtskheta at this time, which has been tentatively attributed to contact with Eurasian nomadic-pastoralists from the steppe of southern Russia. Stable Isotope analysis of archaeological human and faunal remains can provide insight into the diet composition and mobility of people from ancient times. This research examines carbon, nitrogen and strontium isotope ratios (d13C, d15N, and 87Sr/86Sr) of humans excavated from Greater Mtskheta cemeteries remains dating between 1500 BC-AD 700. Diet was compared between time periods, sites, demographic groups (age-at-death, sex) and cultural groups (burial types, modified/unmodified skulls) to illustrate how dietary access or preferences differed over time and between these groups. Further, analyses were used to examine the role migration played in the onset of cultural changes after the 4th century. d13C and d15N results show Greater Mtskheta residents consumed a mixed C3 and C4 diet in the LBA-EIA, which is consistent with a trend in similar studies of human populations in Inner Asia and the South Caucasus during this time. The human diet in Mtskheta transitioned to a C3-dominated diet by the RLA Period, a trend which was also displayed by 87Sr/86Sr populations in the Kislovodsk basin (North Caucasus). LBA-EIA diets differed between men and women, possibly indicating greater animal product consumption among males of this period; while diets became isotopically homogeneous between sites and demographic groups in the RLA period, at a time when social stratification and complexity in Mtskheta was at its peak. The 87Sr/86Sr results demonstrate some individuals with intentionally modified skulls immigrated to Mtskheta after the 4th century, and these individuals may have originated from the vicinity of the North Caucasus or Alazani Valley. This study demonstrates the Greater Mtskheta human diet changed significantly between the LBA-EIA and the RLA with increasing social complexity, and indicates the Greater Mtskheta inhabitants maintained cultural connections with the surrounding regions throughout these time periods, apparently mirroring regional isotopic trends in diet, and evidently accommodating migrants after the 4th century AD.
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ItemInvestigating oligodendrocyte population expansion in the central nervous system( 2022)The myelin sheath is an essential component of central nervous system (CNS) health, without which neuronal function is compromised. Oligodendrocytes are the myelinating cells of the CNS and are produced throughout life through the division and differentiation of oligodendrocyte precursor cells, or OPCs. Interestingly, the processes driving oligodendrocyte production appear distinct between humans and mice. Human OPCs divide quickly and stop dividing with age. There is little oligodendrocyte addition to human white matter tracts in adulthood, and few pre-existing oligodendrocytes are replaced by new cells. By contrast, murine OPCs divide slowly, continue generating new oligodendrocytes in adulthood, and evidence suggests oligodendrocyte replacement is high in adult murine white matter. For these reasons, the relevance of using mice to study human conditions in which oligodendrocytes and myelin are compromised–such as multiple sclerosis (MS)–has recently been questioned. This presents a fundamental hurdle to performing translatable basic research into demyelinating diseases like MS. Oligodendrocyte addition is also required to facilitate new learning, in a phenomenon known as ‘adaptive myelination’. Therefore, distinct oligodendrocyte production dynamics in the human versus the mouse challenges whether studies of murine learning are of clinical relevance to the human. This has severe implications for pre-clinical animal studies which attempt to mitigate the effects of cognitive decline. In this thesis, I use novel techniques to show that both human and mouse OPCs divide quickly and stop dividing with age. I show humans and mice have similar profiles of oligodendrocyte integration over life, and that oligodendrocyte replacement is low in mice as it is in humans. I mount the argument that previous distinctions between human and mice are primarily driven by a tendency to use relatively young mice to assess ‘adult’ white matter change, in conjunction with a tendency to use density-based metrics, rather than measuring total numbers of oligodendroglia. Finally, I investigate whether oligodendrocyte production or survival alters in two established murine models of adaptive myelination: social isolation and environmental enrichment. I find that juvenile social isolation does not alter the course of oligodendrocyte production nor survival in the prefrontal cortex, but that juvenile environmental enrichment transiently increases oligodendrocyte survival. Consistent with recent studies, this suggests that environmental interventions have an exciting potential to promote more efficient modes of oligodendrocyte addition in the CNS. Importantly, this thesis realigns mouse and human oligodendrocyte growth systems to provide credence to the translatability of mouse studies. While the number of oligodendrocytes may be plastic in juvenile development, there is little ability for extensive cellular remodelling in adulthood. This brings forth important questions such as: are there limits to adaptive myelination with ageing? Are there limits to myelin repair? As the human population experiences an increase in the average lifespan, investigating such questions will become key to our ability to therapeutically target white matter repair in neurodegenerative conditions, or preserve cognitive function in age-related cognitive decline.
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ItemThe role of microglia in regulation of vasculature and blood flow in the healthy and diabetic retina( 2022)Diabetic retinopathy is a common vascular complication of diabetes and a leading cause of blindness in those of working age. Prior to overt vascular pathology, the retina displays subtle changes to neurons, glia, and blood vessels that are likely important for disease progression. However, current treatments for diabetic retinopathy are only effective at targeting late-stage pathology. Treatments that target the early cellular changes in the diabetic retina have the potential to halt disease progression before vision is threatened. One of the earliest changes observed in the diabetic retina is a reduction in blood flow. This early vascular dysfunction has been observed in the absence of any other signs of retinopathy, suggesting it may be a key early driver of disease and a promising target for intervention. It has been suggested that the underlying cause of reduced blood flow is dysfunction of the mechanisms that regulate blood flow in the retina. However, our current understanding of these mechanisms is largely incomplete. The central aim of this thesis was, therefore, to explore how blood flow is regulated in the normal retina, and to determine how this function is altered in the diabetic retina. Recent work from our group and others have identified that microglia, the resident immune cells of the central nervous system, may play a role in regulation of blood flow. Based on this emerging evidence, our hypothesis was that microglia regulate vascular function in the retina, and that hyperglycaemia leads to changes in microglia that impair this function and result in reduced blood flow. To explore this hypothesis, we first performed RNA sequencing of retinal microglia isolated from mice lacking Cx3cr1, a chemokine receptor specific to microglia and an important regulator of many microglial functions. This revealed a role for Cx3cr1 in several possible functions related to vasculature, including vascular development, microglial-vascular adhesion, and vascular tone, which were further assessed with in vitro and in vivo imaging techniques. Imaging data revealed the Cx3cr1null retina showed increased vascular density, reduced microglial-vascular contact, and most interestingly, dilation of capillaries. This loss of vascular tone may have been due to reduced expression of angiotensin converting enzyme, a component of the renin angiotensin system (RAS), which promotes vasoconstriction. The ability of microglia to dynamically alter blood vessel diameter and hence control blood flow was then assessed by live cell imaging of the ex vivo retina. We observed frequent spontaneous calcium transients in microglia which appeared to induce vasoconstriction, which may have been mediated by purinergic signalling. Microglia also evoked vasoconstriction via a calcium-independent mechanisms, which was promoted by addition of fractalkine, the ligand for Cx3cr1. Transcriptomic data suggested FKN-Cx3cr1 signalling may promote vasoconstriction via modulation of the RAS. This was confirmed by inhibition of the RAS in the ex vivo retina, which abolished FKN-evoked vasoconstriction. As earlier work from our group has shown FKN-Cx3cr1 signalling and the microglial RAS are upregulated in the diabetic retina concurrent with reduced blood flow, we postulated that this vascular dysfunction may be caused by aberrant microglia-mediated vasoregulation. To test this, we trialled pharmacological blockade of the RAS in an animal model of type 1 diabetes. Without treatment, diabetic animals exhibited constriction of retinal capillaries, reduced blood flow, and dysfunction of inner retinal neurons. Microglia did not display classical signs of activation but did show increased accumulation on capillaries. RAS blockade successfully restored capillary diameter in the diabetic retina, but surprisingly failed to improve blood flow or neuronal function. Finally, while RAS blockade did not affect the number of microglia accumulating on capillaries, it did increase the extent to which individual microglia contacted vasculature, further alluding to the importance of the microglial RAS in regulation of retinal vascular function. In summary, our findings indicate that microglia and Cx3cr1 are important for vascular function in the retina, in particular for vascular development and maintenance of capillary tone. We also established that microglia can dynamically alter blood vessel diameter in multiple ways, suggesting these cells may be important for regulating retinal blood flow. Finally, restoration of capillary diameter by RAS blockade in the diabetic retina supports the theory that aberrant microglia-mediated vasoregulation contributes to early vascular dysfunction in diabetic retinopathy. These findings may form the basis for new treatments that can prevent vascular dysfunction in diabetic retinopathy and other CNS diseases.
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ItemDistinguishing features and regulatory roles of 5-HT containing enteroendocrine cells( 2022)Enteroendocrine cells (EEC) have important roles in communicating the state of the gastrointestinal (GI) tract to the rest of the body, and in signalling within the GI tract. They signal in response to nutrients and metabolites in the GI tract, potentially toxic compounds and mechanical forces. The most numerous EEC signal through 5-hydroxytryptamine (5-HT; also known as serotonin). These were also the first EEC to be identified, which was through their reactions with chrome salts, from which they gained the name enterochromaffin (EC) cells. It was at first thought that all 5-HT producing EEC were much the same. In the recent years, it has become apparent that there are functionally diverse subtypes of 5-HT containing EEC. EEC make up only approximately 1% of the intestinal epithelial cells but they are responsible for producing more than 20 peptide hormones. Of the 1% EEC population, EC cells are the most abundant cell type. I have shown that 5-HT containing EEC co-express a variety of gut hormones (Chapter 2), and their co-expression patterns are defining features that characterise them into various subpopulations based on the hormones they produce. Furthermore, EEC subpopulations express a collection of receptors that respond to different stimuli which impact their physiological effects, thus adding another layer of complexity when classifying the functional subtypes of EEC. 5-HT cells are generally depicted to be open flask-shape cells in the literature. However, one study showed an intriguing characteristic of long basal processes exhibited by some EC cells, though no study had characterised the distinct morphology of 5-HT cells in detail. Therefore, I have undertaken extensive investigations to document the morphological characteristics of 5-HT cells from the mouse stomach to rectum (Chapter 3 and Appendix A). Approximately 50% of 5-HT cells in the mouse distal colon had long basal processes, and this morphology was also observed in the gastric antrum and the rectum. These processes can reach 100 micron in length, and the abundance of this structure must serve some functional roles in the intestinal mucosa. I speculate on these in Chapters 3 and 7, and in Appendix A. An unanswered question arising from the complexity of hormone co-expression is whether co-expressed hormones could be differentially released. To address this, I examined the subcellular distribution of 5-HT and tachykinin (TK) storing secretory vesicles within the same EEC (Chapter 4). 5-HT and TK are stored in separate vesicles, and the two pools of vesicles were preferentially translocated when stimulated with glucose. In addition, duodenal 5-HT/TK cells responded differently than colonic 5-HT/TK cells under the same stimulated condition, suggesting a regional difference of EEC subpopulations. Insulin-like peptide 5 (INSL5) is co-expressed with GLP-1 and PYY in colonic L cells, and I discovered that some 5-HT cells had an intertwining relationship with L cells in the mouse large intestine (Chapter 3). In Chapter 5, I describe the development of an LC/MS assay for an INSL5 analogue that I used to investigate the role of INSL5 (Appendix B). I also discovered that RXFP4, a natural receptor for INSL5, is extensively expressed by colonic 5-HT cells and by some sensory nerve fibres in the mucosa, submucosa, and the muscle layers of the large intestine (Chapter 6). Hence, INSL5 could have an effect on both RXFP4 expressing nerve terminals and on neighbouring 5-HT cells. The regulatory role of 5-HT in the control of colorectal propulsion was demonstrated to be through an INSL5/RXFP4/5-HT/5-HT3R neuro-endocrine circuit. Collectively, my studies presented in this thesis have systematically defined the distinguishing features of 5-HT containing EEC throughout the GI tract and the regulatory role of 5-HT in colonic motility.
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ItemNo Preview AvailablePost-translational control of cell fate decisions: The role of NEDD4 in gonad development( 2022)Gonadogenesis is the process wherein two morphologically distinct organs, the testis and the ovary, arise from a common precursor. In mammals, testicular fate is determined by the expression of Sry. SRY subsequently upregulates the related family member Sox9 which is responsible for initiating testis differentiation while repressing factors critical to ovarian development such as FOXL2 and beta-catenin. The neural precursor cell expressed developmentally down-regulated protein 4 (NEDD4) is a ubiquitin protein ligase that has been implicated in a wide array of cellular processes and the development of many organ systems. In the first part of this thesis (Chapter 3) I characterised the phenotype of XY Nedd4 deficient mice, revealing that, in the absence of NEDD4, XY fetuses exhibit complete male-to-female gonadal sex reversal owing to a reduction in gonadal-precursor cell proliferation, an early failure to initiate the testis-determining program and ectopic expression of, the ovarian transcription factor, FOXL2. This sex reversal extended to germ cells with ectopic expression of SYCP3 in XY Nedd4-/- germ cells and significantly higher Sycp3 transcripts in XY and XX Nedd4 deficient mice when compared to both XY and XX controls. Utilising conditional loss of function approaches, I then explored the cell autonomous roles of NEDD4 in the developing testis (Chapter 4) and ovary (Chapter 5). In doing so I found that, while conditional ablation of Nedd4 was unable to recapitulate the sex reversal observed in XY Nedd4-deficient mice, Nr5a1-Cre;Nedd4flox/flox testes were significantly smaller than littermate controls at all postnatal stages analysed had reduced steroidogenic capacity, owing to a reduction in Lhr and Cyp17a1, resulting in a reduction in seminal vesicle weight and anogenital distance. NEDD4 was found to be largely dispensable for ovarian development, however, although Dazl was increased upon ablation of Nedd4 in XX gonadal somatic cells. In contrast, a drastic phenotype was observed in the adrenal glands of XX Nr5a1-Cre;Nedd4flox/flox mice, which displayed adrenal dysgenesis, resulting in a reduction in adrenocortical cells. In summary, the work within this thesis establishes an essential role for NEDD4 in mammalian sex determination and gonad development and describes hitherto uncharacterised roles for this ubiquitin ligase in murine development.
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ItemComplex spatial 3D anatomy of the tarsal tunnel and the plantar compartments of the foot( 2021)Anatomical understanding of the foot, to a level appropriate to inform precise clinical management, is lacking. Whilst much of the foot is in need of a clear evidence-base, the tarsal tunnel and plantar compartmentalisation of the foot are areas of particular concern for two reasons. First, the anatomy of these areas is readily visualised in a lab setting, so should be well- investigated, yet it is not. Secondly, conditions that affect these areas, such as tarsal tunnel syndrome and plantar compartment syndromes, are common and can be debilitating or even life-threatening. The clinical identification and management of these disorders is varied, has high rates of failure and high rates of patient dissatisfaction from procedures that the clinical team deem successful. Something is amiss, and the confusion and inconsistency in the reviewed literature suggests that imprecise anatomical knowledge may be a prominent contributing factor. The studies presented in this thesis aimed to explore, in sequence, the tarsal tunnel, followed by the medial and lateral columns of the foot. This sequence follows the structures coursing into the plantar aspect of the foot via the tarsal tunnel. These studies aimed to present a detailed anatomical account of these areas by using clear protocols, which involved a variety of dissection, 3D modelling, sectional anatomy and medical imaging techniques. Dissection involved following individual fascicles of soft tissue structures, identifying their orientation and attachments. Each structure was modelled during dissection, as to revaluate the spatial relationships of structures in 3D space post-dissection. Sectional anatomy and medical imaging were used to correlate the dissection findings for what is more readily applied in a clinical setting. The tarsal tunnel roof was described variably in the reviewed literature. The current data demonstrated that the roof is not formed by a singular structure, but through the combination of the passive flexor retinaculum and the dynamic abductor hallucis muscle. The boundaries of the tunnel were defined by the roof, making the description of these tissues of greater importance clinically. The tunnel was also subdivided by fibrous tissues, suggesting that the neurovasculature could be compressed by various surrounding structures. This contrasts much of the reviewed literature and provides anatomical evidence for some failures of surgical release. These data may inform tarsal tunnel surgical care and could increase the effectiveness of these procedures. The plantar aspect of foot was described in the reviewed literature as having anything from zero to ten compartments. Differences in terminology, investigative medium and methodology may have contributed to the variability. The data presented here clearly and consistently demonstrated four distinct compartments of the mid- to hind-foot region: medial, lateral and two central plantar compartments. They had well-defined anatomical boundaries formed by fibrous and muscular intermuscular septae. The relationship of the bounding structures to the muscular and tendinous structures, and the course of the plantar neurovascular bundles was demonstrated digitally in three dimensions. These models may help inform the diagnosis of causative relations for nerve impaction or vascular blockage which lead to compartment syndromes, or simply increase the likelihood of rapid decline in health, such as with foot ulceration in a patient with advanced diabetes. Lower limb amputation and death are common acute sequelae of inadequate treatment of patients in such situations, so improved anatomical understanding may hasten the diagnosis. The studies presented in the form of this thesis provide a technical foundation for future anatomical studies in the foot and other regions of the body. These data provide accurate and reproducible accounts of the complex anatomy of the tarsal tunnel and plantar foot compartments and may have a positive impact on the future care of common foot disorders.
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ItemBenchmarking and developing human pluripotent stem cell models of macrophage biology( 2021)Macrophages are innate immune cells that are found resident in every tissue, and are not only important for host defense, but are also involved in tissue homeostasis, injury, and disease. The advent of human pluripotent stem cells (hPSCs) provides new opportunities to model human macrophages. However, a deeper appreciation of the functional diversity of macrophages, including contextual responsiveness, and innate memory is currently missing from stem cell differentiation studies. Immune responsiveness to pathogenic challenge is known to be impacted by a macrophage's history of prior exposure, but this has not been evaluated in the context of hPSC-derived myeloid cells. This thesis describes the development of a transcriptomic atlas, the Myeloid Atlas, to assess impact of macrophage ontogeny, experimental treatment, and tissue residency on molecular phenotype. In doing so, we have revealed gaps in hPSC-macrophage models, revealing several differences between hPSC-macrophages, peripheral blood monocyte-derived macrophages and primary tissue resident cells. These differences include poor maturation in hPSC-macrophages in the absence of priming signals such as IFN, or repeated exposure to LPS. We demonstrate the requirement for priming in hPSC-macrophages and discover the importance of re-stimulation events in shaping macrophage activation. We further assess phenotypic heterogeneity in both peripheral blood monocyte-derived macrophages and hPSC-macrophages using single-cell RNA sequencing. We demonstrate synchronized population responses to LPS activation and further provide evidence for priming in shaping macrophage responsiveness. Overall, these findings highlight that macrophages are shaped by prior activating, or priming, signals which can be recapitulated in the laboratory using re-stimulation events that shape population phenotypes. The outcomes of this work are expected to improve routine macrophage derivation from hPSC sources, as exploitation of the requirement for in vitro macrophage priming provides future opportunities to shape the quality of acute or long-term macrophage responsiveness for diverse applications.
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ItemNanosecond Laser Therapy for the Management of Age-related Macular Degeneration( 2021)Age-related macular degeneration (AMD) is a chronic, progressive disease affecting the central retina and remains the leading cause of irreversible vision loss in developed nations. AMD is a multifactorial disease encompassing a complex interplay between ageing, environmental and genetic risk factors which ultimately lead to chronic inflammation and death of photoreceptors. While the pathophysiology of AMD is still not fully understood, recent advances have highlighted the involvement of the innate immune system and RPE dysfunction to be fundamental to the development and progression of this debilitating condition. As such, therapies that can influence these two systems have the potential to slow progression of disease. Despite advances in the treatment of neovascular AMD, there remains no effective treatment for non-neovascular AMD and importantly, interventions that address the progression from early to sight-threatening late AMD remain limited. The progression to late stages is usually slow, providing a large window of opportunity to intervene. Novel laser treatments, specifically short-duration pulsed lasers such as the subthreshold nanosecond laser (SNL), offer a potential therapeutic option. The fundamental aims of this study were to investigate SNL-induced cellular response in the RPE that may have potential benefits for addressing pathologic changes that occur in the RPE/BM interface in AMD, and to determine whether changes to the RPE are associated alterations to systemic innate immunity. SNL treatment, delivered in a single session, to the posterior eye in wildtype mice was confirmed using immunohistochemistry to selectively target the RPE without causing damage to the overlying retina. The laser-ablated RPE monolayer was healed within 3 days post-treatment with evidence indicative of newly proliferated daughter cells. Importantly, this proliferative response was also observed in the non-laser treated fellow eye. RNA sequencing and pathway analysis of laser-treated RPE identified biologically-relevant pathways including the promotion of cell survival and cell proliferation, and the inhibition of inflammation and apoptosis. Together, the fellow eye effect and RNA sequencing results suggest that a SNL-induced systemic change involving innate immunity may likely be involved. Systemic change in leukocyte phagocytosis function following SNL treatment was examined for 12-month old wildtype and in the mouse model of AMD (P2X7-null mice). Real-time flow cytometry showed a laser-induced reduction in total phagocytosis capacity of yellow-green fluorescent beads 3 months following SNL treatment. Subsequent ultrastructural studies in the P2X7-null mice showed a significant thinning of the pathologically thickened Bruch’s Membrane (a hallmark feature in AMD). The role of SNL in altering the systemic response, including phagocytosis and cytokine changes, was investigated in a cohort of patients with intermediate AMD. Similar to the rodent results, a substantial reduction in peripheral monocyte phagocytosis was also observed for all subsets of monocyte populations occurring from 3 months following SNL treatment. This reduction was sustained for at least 12 months post-treatment without any significant recovery to pre-laser levels. Cytometric bead array demonstrated significant changes to cytokine-profile with an acute elevation of 9 to 13 cytokines involved in inflammation (of a total of 13 cytokines) at the 2 weeks to 3 month post-treatment time points using 200 SNL spots. At the 6 month time point, a significant increase in interleukin-6 remains in the laser-treated participants compared to pre-laser levels. In summary, these findings indicate that SNL can induce a rejuvenating response by promoting RPE proliferation in both the treated eye and the non-treated eye while also reducing the thickness of BM in the eye that received SNL treatment. This study also provides evidence that SNL therapy has the potential to modify systemic immune responses, including phagocytosis function and cytokine-profile. Taken together, our results indicate a potential role for SNL treatment of AMD to slow progression of disease.
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ItemInvestigating regulation of vagally-evoked respiratory responses by the parabrachial complex in the mouse( 2022)The presence of respiratory irritants or infection can promote aberrant activity of central nervous system (CNS) neural circuits, which conduct interoceptive and homeostatic activity. Dysfunctional action in these CNS circuits contribute to morbidity in disease and decrease in quality of life. Incomplete understanding of CNS respiratory sensory relay circuits likely precludes effective medical treatment in instances of aberrant neural activity. Thus far, our understanding of respiratory relay aspects of the parabrachial complex (PBC) is unassessed. The PBC has been known to relay pain, thermoregulatory, gustatory, fluid and salt intake, and other sensory inputs to forebrain regions. Additionally, the PBC is known to receive respiratory-relevant sensory information to its pontine respiratory group (PRG) division, which in turn regulates respiratory pattern. However, the potential of the PBC in relaying vagally-derived respiratory-relevant information to forebrain regions has not been assessed. This thesis investigated physiological, neurochemical, and neuroanatomical components of the PBC in mice to assess its role in relaying vagal sensory information. To achieve this, a novel methodological approach was developed to facilitate physiological investigation of changes in respiratory rate, in response to electrical stimulation of the vagus nerve (eVNS), combined with muscimol inhibition of select PBC areas. Investigation identified region-dependent modulation of eVNS-induced activity by the PBC, with distinct roles for the medial parabrachial (MPB) and lateral parabrachial (LPB). Muscimol microinjection into the caudal LPB resulted in the complete abolishment of eVNS-induced apnoea. Inhibition of the caudal or intermediate MPB potentiated eVNS-induced tachypnoea, with inhibition of intermediate MPB regions additionally increasing the eVNS frequency required to induce apnoea. Given these physiological findings, the neurochemical phenotypes and neuroanatomical connectivity of the PBC were next investigated with immunohistochemistry and viral tracing, with an emphasis on description of the caudal PBC, a less understood subregion. The immunohistochemical findings obtained here suggest that the caudal LPB appears to be a unique entity, distinct from the intermediate and rostral LPB, due to the absence of CGRP or ChAT somal labelling in the caudal LPB which was present in the intermediate and rostral LPB. Additionally, calbindin immunolabelling identified a previously undescribed ‘axonal bundle’ principally present in caudal PBC regions. Viral tracing used here did not allow a discrete investigation of the caudal PBC and its connectivity in isolation, yet replicated projection patterns of the PBC that had previously been comprehensively assessed only in rats. Overall, this thesis has identified the caudal PBC to be a novel region of vagal sensory relay, deserving of future investigation.
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ItemThe Clinical Anatomy of the Hallucal Sesamoids and their Attachments( 2021)The hallucal sesamoid bones are essential, but poorly understood, features of the first, or great, toe complex. With the associated structures of the foot, collectively termed the first ray, this complex is essential for optimal foot function. Disorders of this region present symptomatically in a broad range from intermittent discomfort through to a complete inability to walk or weight-bear. Pain in this region may also be enough to interrupt sleep and a range of daily tasks. Given the perceived importance of the hallucal sesamoids to this complex, it is perhaps surprising to discover the anatomy of the hallucal sesamoids, and particularly their interaction with neighbouring tissues, is poorly understood. One major consequence of this is the lack of a clear, consistent anatomical foundation for the diagnosis, treatment (including surgically) and management of these disorders. This thesis will document a series of studies which aim to collectively enhance the anatomical understanding of the hallucal sesamoids and their relations. After exploring the historical context of the hallucal sesamoids, reviewing the current anatomical and clinical literature, and establishing some key concepts about the presence, absence and variability of these bones, a series of detailed studies will be presented. Using a multi-modal approach that includes dissection, 3D modelling, medical imaging and sectional anatomy (macro and micro), the key tissues thought to interact with the hallucal sesamoids will be investigated: the adductor and abductor hallucis muscles, the plantar aponeurosis, the flexor hallucis brevis muscle and a range of highly specialised ligaments. The ligaments include the deep transverse metatarsal and sesamophalangeal, , amongst others, along with interactions with other tissues such as tendon tunnels, plantar plates and joint capsules. Of particular importance when considering the function of these structures is their attachment to skeletal tissues, such as the hallucal sesamoids. There is contention over what tissues are attached to each of the two hallucal sesamoids. These studies provide a detailed, quantified and uniquely visualised account of the attachments to each bone, and to neighbouring structures. This comprehensive account provides a strong basis for the development of more anatomically informed diagnostic approaches and clinical management of disorders of this area. The key findings presented in this thesis include definitive three dimensional mapping of the detailed attachments to each hallucal sesamoid. These dispel common misconceptions that the sesamoids are engulfed by a single tissue, or that they do not have substantial connections to a range of structures. These data strongly demonstrate these sesamoids have multidirectional interactions which maintain their dynamic position through the gait cycle, but are also particularly vulnerable to injury, including iatrogenic injury. In fact, poor post-operative outcomes from surgical treatments in this area may be attributable to a lack of anatomical understanding. Studies demonstrate the intimate relationships between structures often targeted for surgical release and those which must be maintained. These relations, and particularly the innovative three-dimensional visualisations developed from these data, may inform significant advances in first ray health care.