115 research outputs found
An assessment of small vessel disease in human post-mortem tissue through radiology and histology
Sporadic human cerebral small vessel disease (SVD) is a significant problem in our aging population. It is the most common cause of haemorrhagic stroke, causes one quarter of all ischaemic strokes, causes vascular dementia and synergistically worsens other dementias. It also causes a range of other psychological and physical problems and is increasingly common with increased age. SVD is well characterised on neuroimaging, with lesions throughout the brain, and particularly in the white matter. The cause(s) and pathophysiological mechanisms underlying the development of SVD, however, remain poorly understood with poor correlations between the abnormalities seen at the cellular level, on neuroimaging, and clinically.
There are many theories as to the cause of SVD. However, observational studies and experimental evidence point towards an abnormality in the small vasculature of the brain initiating a cascade of events leading to a variety of vascular and brain parenchymal lesions. What these abnormalities are, and how exactly they result in the pathology seen, is unknown. Different structural components of the vessel wall and parenchymal brain cells appear to be involved, as well as functional abnormalities such as abnormal vascular reactivity. Risk factors also play a role, hypertension being the most significant, but how these interact with the normal vasculature is not fully understood.
To provide an overview of our current understanding of SVD in human tissue I first completed a systematic review of the literature comparing the appearances of SVD on post-mortem imaging and histology. This revealed the inconsistency in methods and reporting in these studies and the lack of histopathology agreement on SVD terminology and definitions.
I then studied the histological appearances of the lesions identified by post-mortem imaging to provide a reliable precise histological-imaging correlation. I developed a new protocol for ex vivo 7 Tesla magnetic resonance imaging (7T MRI) scanning of human brain tissue on post-mortem material and developed a grading system to assess SVD burden on MRI and histology with histological definitions, to try to encourage standardised, comprehensive and transparent reporting so that results in small studies can be more easily compared. I studied human post-mortem brain tissue to better investigate the disease in the appropriate context.
In our cases from individuals with haemorrhagic SVD, normal aging and young controls, the most severe SVD pathology on ex vivo imaging and histology was, as expected, in the haemorrhagic SVD group. The normal aging group also had significant levels of pathology, perhaps representing the increasing burden of disease present but not necessarily detected clinically with increased age. It is possible the underlying pathophysiology in this group might develop by different mechanisms compared to the haemorrhagic group. Directly comparing the imaging and histological lesions confirmed the histological appearances of some lesions on imaging such as enlarged perivascular spaces, lacunes, microinfarcts and microbleeds. However, making direct comparisons is complex. Some lesions, such as small vessel fibrinoid necrosis, presumed to be below the resolution for detection on 7T MRI, were identified on both histology and imaging. Some features seen on histology in association with recognised SVD lesions, such as perivascular inflammation in an area of white matter rarefaction, were present in a variety of different histological contexts with no apparent correlation on imaging. And some lesions, such as white matter rarefaction around enlarged perivascular spaces, were present often on both imaging and histology, but their significance and contribution to SVD is unknown.
To try to further understand the mechanisms underlying SVD and the lesions seen on imaging I undertook biochemical studies of protein expression in the deep white matter of the haemorrhagic SVD group, young controls and an Alzheimer’s disease group, who also have white matter pathology on neuroimaging. Increased fibrinogen levels suggested vascular leakage in both disease groups. However, haemorrhagic SVD had more severe white matter hypoxic changes and increased vasoconstrictor levels while in Alzheimer’s disease there was increased amyloid 42 and levels of a pericyte marker, possibly reflecting different pathophysiological mechanisms causing the similar appearing radiological changes.
When assessing radiologically defined white matter hyperintensities (WMH) I found hypoxic-induced changes throughout brains with WMH, including in normal appearing areas of white matter. This suggests these brains have abnormalities in areas that appear radiologically normal, as found in in vivo imaging studies.
To conclude, this work has confirmed the importance of reaching a consensus in histopathological reporting, terminology and definitions which is a basic requirement before we can better understand the pathophysiology of SVD. This has led to the formation of definitions and a practical grading system that could be used as a basis upon which to build a future agreement. The complexity of the histological lesions underlying radiological SVD changes was apparent, and the frequency with which some other potentially important histological changes were identified suggests these have not, to date, been fully appreciated. Investigating the underlying mechanisms of white matter hyperintensities showed vascular leakage was a shared abnormality in two different diseases with white matter changes on imaging, suggesting it may be a common factor upon which variable pathways converge. Future work is needed to further understand the importance of these less well characterised histological features. Investigating the role of vascular leakage and exploring drugs that maintain or improve vascular integrity could be a potential route for helping to treat SVD. Studies into underlying transcriptomic abnormalities around vascular leakage in human tissue may be informative
Characterisation of the Sensory Phenotype of the Oesophageal Mucosa in Adults with Gastroesophageal Reflux Disease
As the first line of defence against noxious luminal contents, the oesophageal mucosa plays an important role in the pathogenesis of gastroesophageal reflux disease (GORD). We hypothesised that the heterogeneity of symptom perception between heartburn patients may be dependent on the interactions between neuronal and inflammatory cells in the oesophageal mucosa. Studies were conducted with endoscopic oesophageal biopsies from GORD patients (N=83) to characterise the phenotype of afferent mucosal nerve endings by assessing expression of ion channels TRPV1, ASIC3, and TRPM8 involved in pain transduction. Neuro-immune interactions in the oesophageal mucosa of heartburn patients and healthy individuals (N=14) was studied by assessing the expression of inflammatory cytokine receptors CXCR2, TNFR1, IL1R, IL6R, and RAMP1. Infiltration of immune cell populations was characterised among GORD groups. Mast cell co-expression of NGF was also studied. Cytokine release profiles were measured in supernatant from mucosal biopsies from ERD patients and healthy controls exposed to acid using a multiplex assay. RNA extracted from mucosal GORD biopsies and healthy controls were bulk-sequenced to assess the differences in the molecular gene signature between patients with heartburn and asymptomatic subjects, and among GORD phenotypes. TRPV1 was expressed only on mucosal superficial sensory afferent nerve endings of NERD patients, while ASIC3 was most frequently expressed on oesophageal epithelial cells in NERD and ERD patients. CXCR2 was localised on epithelial cells surrounding the papillae in all GORD phenotypes and healthy controls, but was also detected on deep sensory afferent nerves innervating papillae in FH patients. NGF expression was significantly higher in mast cells in patients with GORD compared to healthy controls, and these mast cells were detected in close proximity to sensory deep afferent nerves in patients with ERD. IL8 secretion was increased with acid exposure in both healthy control oesophageal mucosa and ERD, while NGF was released at higher levels with acid exposure from ERD oesophageal mucosa. RNA sequencing detected important differences in expression of genes with structural and regenerative roles between NERD, ERD and BO oesophageal mucosa and asymptomatic subjects. We demonstrated distinct sensory phenotypes in the oesophageal mucosa of patients with ERD, NERD, BO, FH, and healthy controls. These phenotypes include differences in mucosal afferent innervation, immune cell profiles, cytokine release when challenged with acid, and gene expression signatures. Collectively, these findings may contribute to heartburn pathogenesis in the oesophageal epithelium of GORD patients. Improved understanding of mucosal targets identified in this study, including TRPV1, ASIC3, and NGF, in functional studies will enable development of targeted topical treatments to alleviate heartburn symptoms
A Meeting of Minds: In Recognition of the Contributions of Randall J. Cohrs
A Special Issue in memory of Randall J. Cohrs, Ph.D. Topics include original research reports on a variety of viruses as well as reviews and commentaries on Randy’s contributions to many investigations
Human Papillomavirus and Related Diseases
Cervical cancer is the second most prevalent cancer among women worldwide, and infection with Human Papilloma Virus (HPV) has been identified as the causal agent for this condition. The natural history of cervical cancer is characterized by slow disease progression, rendering the condition in essence preventable and even treatable when diagnosed in early stages. Pap smear and the recently introduced prophylactic vaccines are the most prominent prevention options, but despite the availability of these primary and secondary screening tools, the global burden of disease is unfortunately still very high This book will focus on the clinical and diagnostic aspects of HPV and related disease, highlighting the latest developments in this field
Bioengineering Functional Human Jejunal Grafts for Intestinal Failure
Intestinal failure (IF), following extensive anatomical or functional loss of small intestine, has debilitating long-term effects on infants born with this condition. Priority of care is to increase the child’s length of functional intestine, jejunum in particular, to improve nutritional independence. Children with irreversible IF suffering complications of parenteral nutrition may be referred for intestinal transplantation. However, mortality rates are as high as 60% at 5 years. The aim of my project was to develop an innovative treatment strategy to rebuild the patients’ own jejunum for autologous transplantation. Here I report the reconstruction of transplantable intestinal mucosal grafts using primary human materials. Human jejunal intestinal organoids derived from paediatric patients with IF can be cultured and expanded efficiently in vitro with region-specific markers preserved. Decellularised human intestinal matrix with intact ultrastructure is used as biological scaffold. I show that the biochemical composition of decellularised human small intestine and colon matrix are virtually analogous, suggesting that they both can be used as scaffolds for jejunal graft reconstruction. Functional jejunal grafts with digestive enzyme-producing enterocytes can be efficiently engineered by repopulating human intestinal scaffolds with human jejunal organoids and fibroblasts in vitro, which can further survive and mature after in vivo transplantation. These primary human material based jejunal grafts provide proof-of-concept data for autologous transplantation of tissue engineered intestine in patients with IF
Embryonic Stem Cells
Embryonic stem cells are one of the key building blocks of the emerging multidisciplinary field of regenerative medicine, and discoveries and new technology related to embryonic stem cells are being made at an ever increasing rate. This book provides a snapshot of some of the research occurring across a wide range of areas related to embryonic stem cells, including new methods, tools and technologies; new understandings about the molecular biology and pluripotency of these cells; as well as new uses for and sources of embryonic stem cells. The book will serve as a valuable resource for engineers, scientists, and clinicians as well as students in a wide range of disciplines
Development of a high-throughput drug screening platform for oligodendrocyte myelination (for progressive multiple sclerosis)
Aim
As part of the broad strategy in Edinburgh and beyond to discover new treatments for
progressive Multiple Sclerosis (MS), the aims for my PhD project were: (1) to address the lack
of an in vitro phenotypic drug screening platform that is able to fully recapitulate myelin
sheath formation, with the long-term goal being to enhance the discovery of pro
remyelination therapies in progressive MS, and target the poor drug discovery rates in brain
disorders which is partially due to poor disease modelling, and (2) the exploration of non
linear optical imaging microscopy techniques that targets both the resolution and speed to
study myelination.
Background
In multiple sclerosis, an inflammatory autoimmune process destroys the oligodendrocytes
that provide neuronal support by forming multi-layered compact myelin sheaths around
axons, leading to neurodegeneration. Although there are drugs available to suppress the
inflammatory attack to limit the formation of demyelinated lesions, no treatments currently
exist to promote the regeneration of these myelin sheaths once the damage has occurred.
Cell based screens have formed an important part of the strategy used to discover such
regenerative drugs. The majority of published cell-based phenotypic drug screens to target
repair have focused on the differentiation of oligodendrocyte precursor cells into
oligodendrocytes rather than their ability to form mature protective myelin sheaths.
However, in many MS lesions, pre-myelinating oligodendrocytes are present, and studies on
oligodendrocyte biology show that differentiation and myelination are regulated by distinct
mechanisms. There is therefore a need for novel drug screens that target the later
myelinating stages of oligodendrocyte development.
Results
Using a 3D microfibre system for in vitro myelin sheath formation (described by Bechler,
Byrne and ffrench-Constant (2015)), I first asked whether compounds that had been
v
identified as increasing differentiation in conventional 2D culture systems enhanced
myelination in the 3D cultures. While Benztropine and Clemastine showed an increase in the
number of MBP+ (differentiated) oligodendrocytes in the 2D system, consistent with
previous publications, no increase in myelin sheath formation was seen with any of the drugs
in the 3D system, highlighting the potentially important differences between differentiation
and myelin sheath formation for drug discovery.
Next I developed a multiwell plate based assay to allow 3D myelination assays to be used for
drug screening. Using electrospinning to produce PLA microfibres, I was able to develop and
optimise a technique to insert and suspend the fibres across the bottom of a 96-well plate
that can be incorporated into an automated pipeline for high-throughput drug screening. The
3D myelin sheaths could be imaged using the Leica SP8 confocal system with the
MatrixScreener extension and the Opera Phenix high-content screening system.
Finally, I addressed the problem of imaging myelin in such screens. CARS was shown to be
able to preferentially detect myelin sheaths in fixed and live slices in regions and time-points
of varying myelin densities. As the development of new myelin sheaths requires the
formation of lipid and therefore the incorporation of hydrogen, the consumption of D2O (heavy water) with a 2H atom allowed the non-invasive labelling and detection of myelin
sheaths using SRS. Future experiments will allow us to confirm whether this deuterium
detection is preferential and/or specific to new myelin sheaths.
Significance
With only about 10% of drugs that enter Phase I trials successfully launching into clinics, there
is an important need for more effective drug screens that better model disease-relevant
processes and so reduce late-stage failures. The high-throughput-compatible 96-well plate
with suspended PLA microfibres that combines the recent progresses in 3D cellular model
systems with bioengineering and the recent advances in high content imaging systems may
give us the opportunity to more accurately model these disease-relevant structures in vitro,
and therefore improve drug discovery for regenerative therapies in multiple sclerosis and
other myelin diseases.
The research on Raman-based label-free imaging of myelin sheaths is not only applicable for
imaging myelin sheaths in the context of drug validation, but could be important for live imaging of brain slices and detection of newly-formed myelin sheaths without the need for
complex and expensive transgenic animals
Studies of crustacean hyperglycaemic hormone in the Norway lobster 'Nephrops norvegicus' (Linnaeus, 1758)
Nephrops norvegicus is a deep water marine decapod crustacean which burrows in fine muddy substrata. It is a commercially important as a fisheries species, but knowledge of its biology, particularly concerning its endocrinology, is limited. This thesis describes the endocrinology of Nephrops norvegicus with particular reference to crustacean hyperglycaemic hormone. Histological investigations of the eyestalk of Nephrops norvegicus enabled the identification d" the X-organ sinus gland complex. The development of a microbioassay allowed the determination of increases of glycaemia following the injection of crude sinus gland extracts into a host animal. The optimal dose for induced haemolymph glycaemia was determined. HPLC separation was used to isolate and purify several neuropeptides from crude sinus gland extracts, which had varying degrees of hyperglycaemic activities. The use of ELISA (enzyme linked immunosorbent assay) showed that the peptides reacted with polyclonal rabbit antiserum raised against the CHH of the crayfish Orconectes limosus and with polyclonal guinea pig antiserum raised against the CHH of the lobster, Homarus americanus. SDS-PAGE of these peptides enabled an estimation of their molecular weights and the purity of one of the active peptides was determined using capillary electrophoresis. The effects of photoperiod and severe hypoxia on the CHH-induced hyperglycaemia of Nephrops norvegicus were also investigated. The responses of N. norvegicus appeared to differ in some respects from other decapods species. Pairs of oligonucleotide primers, based on the sequence of the lobster, Homarus americanus, complimentary to regions of the CHH sequence, were used in the polymerase chain reactions (PCR). Complimentary first strand DNA (cDNA) was synthesised from total Nephrops eyestalk RNA. 35 rounds and 40 rounds of PCR amplification produced a l00bp and 230bp double stranded DNA product respectively, which were resolved by electrophoresis on a tris-borate-EDTA gel. The product size was compared with known standards. Finally, the identification of CHH and GIH synthesis and storage in the eggs of Nephrops norvegicus at various stages of development was investigated. By the use of PCR, it was not possible to determine if synthesis of CHH was occurring in 50% developed eggs. The use of ELISA, however, demonstrated that in 90% developed eggs there was a significant increase of both CHH and GIH immunoactivity
Investigation into experimental toxicological properties of plant protection products having a potential link to Parkinson's disease and childhood leukaemia
In 2013, EFSA published a literature review on epidemiological studies linking exposure to pesticides and human health outcome. As a follow up, the EFSA Panel on Plant Protection Products and their residues (PPR Panel) was requested to investigate the plausible involvement of pesticide exposure as a risk factor for Parkinson's disease (PD) and childhood leukaemia (CHL). A systematic literature review on PD and CHL and mode of actions for pesticides was published by EFSA in 2016 and used as background documentation. The Panel used the Adverse Outcome Pathway (AOP) conceptual framework to define the biological plausibility in relation to epidemiological studies by means of identification of specific symptoms of the diseases as AO. The AOP combines multiple information and provides knowledge of biological pathways, highlights species differences and similarities, identifies research needs and supports regulatory decisions. In this context, the AOP approach could help in organising the available experimental knowledge to assess biological plausibility by describing the link between a molecular initiating event (MIE) and the AO through a series of biologically plausible and essential key events (KEs). As the AOP is chemically agnostic, tool chemical compounds were selected to empirically support the response and temporal concordance of the key event relationships (KERs). Three qualitative and one putative AOP were developed by the Panel using the results obtained. The Panel supports the use of the AOP framework to scientifically and transparently explore the biological plausibility of the association between pesticide exposure and human health outcomes, identify data gaps, define a tailored testing strategy and suggests an AOP’s informed Integrated Approach for Testing and Assessment (IATA)
Photonic discrimination and specific targeting of vascular stem cells.
Cardiovascular disease remains the leading cause of death and disability world-wide. The current treatment options include balloon angioplasty and the deployment of drug-eluting stents (DES) coated with anti-mitotic drugs to prevent intimal-medial thickening (IMT). Despite this, an unacceptably high failure rate remains due to non-specific targeting of cells and drug-depletion over time. The source of the cells contributing to IMT remains controversial; one theory suggests a reprogramming of native differentiated vascular smooth muscle cells (SMC) while the other proposes myogenic differentiation of resident vascular and/or circulating stem cells. Resolution of this controversy through identification of the source of the contributing cells would greatly assist in the development of future drug targeting strategies using novel DES platforms.
The use of photonics and vibrational spectroscopy is gaining popularity for disease diagnosis. Both platforms have the ability to yield cellular and molecular information about cells and tissues label-free, making them attractive technologies for analysing biological specimen. The first main objective of this work was to analyse individual cells from normal (healthy) and arteriosclerotic (diseased) vessels ex vivo using autofluorescence (AF) in response to broadband light and to compare their AF signatures to undifferentiated stem cells and their myogenic progeny in vitro. The second aim was to use vibrational spectroscopy (Raman and FTIR) to examine undifferentiated stem cells, their myogenic and osteogenic progeny and and further compare their spectra to re- programmed differentiated SMC.
Finally, a novel therapeutic platform for targeting stem cell-derived myogenic progeny using magnetic nanoparticles was developed. Using pharmacological inhibitors of glycogen synthase 3 beta (GSK3β), the effects on Notch, a well known mediator of myogenic differentiation were first evaluated in vitro. Further to this, a prototype GSK3β inhibitor was incorporated into a novel drug delivery system consisting of polymer coated Fe304 magnetic nanoparticles which can be systemically administered and specifically targeted to bare-metal stents by an external magnetic field
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