Developing Multimodal Spectroscopic Imaging Techniques to Study Metal Dyshomeostasis and Altered Brain Biochemistry During Ageing

Cognitive decline and neurodegenerative diseases such as Alzheimer’s disease, pose significant concerns for the ageing population. Increased understanding of the biochemical processes that occur during ageing, may help to reveal pathways for prevention or treatment. There has been interest in the potential role that metal dis-homeostasis plays during ageing and cognitive decline, so this thesis has focussed on developing and applying spectroscopic methods to study metal dis-homeostasis and concomitant biochemical changes during ageing

A review of the “metallome” within neurons and glia, as revealed by elemental mapping of brain tissue

It is now well established that transition metals, such as Iron (Fe), Copper (Cu), and Zinc (Zn) are necessary for healthy brain function. Although Fe, Cu, and Zn are essential to the brain, imbalances in the amount, distribution, or chemical form (“metallome”) of these metals is linked to the pathology of numerous brain diseases or disorders. Despite the known importance of metal ions for both brain health and disease, the metallome that exists within specific types of brain cells is yet to be fully characterised. The aim of this mini-review is to present an overview of the current knowledge of the metallome found within specific brain cells (oligodendrocytes, astrocytes, microglia, and neurons), as revealed by direct elemental mapping techniques. It is hoped this review will foster continued research using direct elemental mapping techniques to fully characterise the brain cell metallome

“Wax On, Wax Off”: In Vivo Imaging of Plant Physiology and Disease with Fourier Transform Infrared Reflectance Microspectroscopy

Analysis of the epicuticular wax layer on the surface of plant leaves can provide a unique window into plant physiology and responses to environmental stimuli. Well-established analytical methodologies can quantify epicuticular wax composition, yet few methods are capable of imaging wax distribution in situ or in vivo. Here, the first report of Fourier transform infrared (FTIR) reflectance spectroscopic imaging as a non-destructive, in situ, method to investigate variation in epicuticular wax distribution at 25 µm spatial resolution is presented. The authors demonstrate in vivo imaging of alterations in epicuticular waxes during leaf development and in situ imaging during plant disease or exposure to environmental stressors. It is envisaged that this new analytical capability will enable in vivo studies of plants to provide insights into how the physiology of plants and crops respond to environmental stresses such as disease, soil contamination, drought, soil acidity, and climate change

Sample preparation with sucrose cryoprotection dramatically alters Zn distribution in the rodent hippocampus, as revealed by elemental mapping

Transition metal ions (Fe, Mn, Cu, Zn) are essential for healthy brain function, but altered concentration, distribution, or chemical form of the metal ions has been implicated in numerous brain pathologies. Currently, it is not possible to image the cellular or sub-cellular distribution of metal ions in vivo and therefore, studying brain-metal homeostasis largely relies on ex vivo in situ elemental mapping. Sample preparation methods that accurately preserve the in vivo elemental distribution are essential if one wishes to translate the knowledge of elemental distributions measured ex vivo toward increased understanding of chemical and physiological pathways of brain disease. The choice of sample preparation is particularly important for metal ions that exist in a labile or mobile form, for which the in vivo distribution could be easily distorted by inappropriate sample preparation. One of the most widely studied brain structures, the hippocampus, contains a large pool of labile and mobile Zn. Herein, we describe how sucrose cryoprotection, the gold standard method of preparing tissues for immuno-histochemistry or immuno-fluorescence, which is also often used as a sample preparation method for elemental mapping studies, drastically alters hippocampal Zn distribution. Based on the results of this study, in combination with a comparison against the strong body of published literature that has used either rapid plunge freezing of brain tissue, or sucrose cryo-protection, we strongly urge investigators in the future to cease using sucrose cryoprotection as a method of sample preparation for elemental mapping, especially if Zn is an analyte of interest

COVID-19 trajectories among 57 million adults in England: a cohort study using electronic health records

BACKGROUND: Updatable estimates of COVID-19 onset, progression, and trajectories underpin pandemic mitigation efforts. To identify and characterise disease trajectories, we aimed to define and validate ten COVID-19 phenotypes from nationwide linked electronic health records (EHR) using an extensible framework. METHODS: In this cohort study, we used eight linked National Health Service (NHS) datasets for people in England alive on Jan 23, 2020. Data on COVID-19 testing, vaccination, primary and secondary care records, and death registrations were collected until Nov 30, 2021. We defined ten COVID-19 phenotypes reflecting clinically relevant stages of disease severity and encompassing five categories: positive SARS-CoV-2 test, primary care diagnosis, hospital admission, ventilation modality (four phenotypes), and death (three phenotypes). We constructed patient trajectories illustrating transition frequency and duration between phenotypes. Analyses were stratified by pandemic waves and vaccination status. FINDINGS: Among 57 032 174 individuals included in the cohort, 13 990 423 COVID-19 events were identified in 7 244 925 individuals, equating to an infection rate of 12·7% during the study period. Of 7 244 925 individuals, 460 737 (6·4%) were admitted to hospital and 158 020 (2·2%) died. Of 460 737 individuals who were admitted to hospital, 48 847 (10·6%) were admitted to the intensive care unit (ICU), 69 090 (15·0%) received non-invasive ventilation, and 25 928 (5·6%) received invasive ventilation. Among 384 135 patients who were admitted to hospital but did not require ventilation, mortality was higher in wave 1 (23 485 [30·4%] of 77 202 patients) than wave 2 (44 220 [23·1%] of 191 528 patients), but remained unchanged for patients admitted to the ICU. Mortality was highest among patients who received ventilatory support outside of the ICU in wave 1 (2569 [50·7%] of 5063 patients). 15 486 (9·8%) of 158 020 COVID-19-related deaths occurred within 28 days of the first COVID-19 event without a COVID-19 diagnoses on the death certificate. 10 884 (6·9%) of 158 020 deaths were identified exclusively from mortality data with no previous COVID-19 phenotype recorded. We observed longer patient trajectories in wave 2 than wave 1. INTERPRETATION: Our analyses illustrate the wide spectrum of disease trajectories as shown by differences in incidence, survival, and clinical pathways. We have provided a modular analytical framework that can be used to monitor the impact of the pandemic and generate evidence of clinical and policy relevance using multiple EHR sources. FUNDING: British Heart Foundation Data Science Centre, led by Health Data Research UK

The adverse impact of COVID-19 pandemic on cardiovascular disease prevention and management in England, Scotland and Wales: A population-scale analysis of trends in medication data

Objectives To estimate the impact of the COVID-19 pandemic on cardiovascular disease (CVD) and CVD management using routinely collected medication data as a proxy. Design Descriptive and interrupted time series analysis using anonymised individual-level population-scale data for 1.32 billion records of dispensed CVD medications across 15.8 million individuals in England, Scotland and Wales. Setting Community dispensed CVD medications with 100% coverage from England, Scotland and Wales, plus primary care prescribed CVD medications from England (including 98% English general practices). Participants 15.8 million individuals aged 18+ years alive on 1 st April 2018 dispensed at least one CVD medicine in a year from England, Scotland and Wales. Main outcome measures Monthly counts, percent annual change (1 st April 2018 to 31 st July 2021) and annual rates (1 st March 2018 to 28 th February 2021) of medicines dispensed by CVD/ CVD risk factor; prevalent and incident use. Results Year-on-year change in dispensed CVD medicines by month were observed, with notable uplifts ahead of the first (11.8% higher in March 2020) but not subsequent national lockdowns. Using hypertension as one example of the indirect impact of the pandemic, we observed 491,203 fewer individuals initiated antihypertensive treatment across England, Scotland and Wales during the period March 2020 to end May 2021 than would have been expected compared to 2019. We estimated that this missed antihypertension treatment could result in 13,659 additional CVD events should individuals remain untreated, including 2,281 additional myocardial infarctions (MIs) and 3,474 additional strokes. Incident use of lipid-lowering medicines decreased by an average 14,793 per month in early 2021 compared with the equivalent months prior to the pandemic in 2019. In contrast, the use of incident medicines to treat type-2 diabetes (T2DM) increased by approximately 1,642 patients per month. Conclusions Management of key CVD risk factors as proxied by incident use of CVD medicines has not returned to pre-pandemic levels in the UK. Novel methods to identify and treat individuals who have missed treatment are urgently required to avoid large numbers of additional future CVD events, further adding indirect cost of the COVID-19 pandemic

Linked electronic health records for research on a nationwide cohort of more than 54 million people in England: data resource

Abstract: Objective: To describe a novel England-wide electronic health record (EHR) resource enabling whole population research on covid-19 and cardiovascular disease while ensuring data security and privacy and maintaining public trust. Design: Data resource comprising linked person level records from national healthcare settings for the English population, accessible within NHS Digital’s new trusted research environment. Setting: EHRs from primary care, hospital episodes, death registry, covid-19 laboratory test results, and community dispensing data, with further enrichment planned from specialist intensive care, cardiovascular, and covid-19 vaccination data. Participants: 54.4 million people alive on 1 January 2020 and registered with an NHS general practitioner in England. Main measures of interest: Confirmed and suspected covid-19 diagnoses, exemplar cardiovascular conditions (incident stroke or transient ischaemic attack and incident myocardial infarction) and all cause mortality between 1 January and 31 October 2020. Results: The linked cohort includes more than 96% of the English population. By combining person level data across national healthcare settings, data on age, sex, and ethnicity are complete for around 95% of the population. Among 53.3 million people with no previous diagnosis of stroke or transient ischaemic attack, 98 721 had a first ever incident stroke or transient ischaemic attack between 1 January and 31 October 2020, of which 30% were recorded only in primary care and 4% only in death registry records. Among 53.2 million people with no previous diagnosis of myocardial infarction, 62 966 had an incident myocardial infarction during follow-up, of which 8% were recorded only in primary care and 12% only in death registry records. A total of 959 470 people had a confirmed or suspected covid-19 diagnosis (714 162 in primary care data, 126 349 in hospital admission records, 776 503 in covid-19 laboratory test data, and 50 504 in death registry records). Although 58% of these were recorded in both primary care and covid-19 laboratory test data, 15% and 18%, respectively, were recorded in only one. Conclusions: This population-wide resource shows the importance of linking person level data across health settings to maximise completeness of key characteristics and to ascertain cardiovascular events and covid-19 diagnoses. Although this resource was initially established to support research on covid-19 and cardiovascular disease to benefit clinical care and public health and to inform healthcare policy, it can broaden further to enable a wide range of research

Linked electronic health records for research on a nationwide cohort of more than 54 million people in England: data resource

Abstract: Objective: To describe a novel England-wide electronic health record (EHR) resource enabling whole population research on covid-19 and cardiovascular disease while ensuring data security and privacy and maintaining public trust. Design: Data resource comprising linked person level records from national healthcare settings for the English population, accessible within NHS Digital’s new trusted research environment. Setting: EHRs from primary care, hospital episodes, death registry, covid-19 laboratory test results, and community dispensing data, with further enrichment planned from specialist intensive care, cardiovascular, and covid-19 vaccination data. Participants: 54.4 million people alive on 1 January 2020 and registered with an NHS general practitioner in England. Main measures of interest: Confirmed and suspected covid-19 diagnoses, exemplar cardiovascular conditions (incident stroke or transient ischaemic attack and incident myocardial infarction) and all cause mortality between 1 January and 31 October 2020. Results: The linked cohort includes more than 96% of the English population. By combining person level data across national healthcare settings, data on age, sex, and ethnicity are complete for around 95% of the population. Among 53.3 million people with no previous diagnosis of stroke or transient ischaemic attack, 98 721 had a first ever incident stroke or transient ischaemic attack between 1 January and 31 October 2020, of which 30% were recorded only in primary care and 4% only in death registry records. Among 53.2 million people with no previous diagnosis of myocardial infarction, 62 966 had an incident myocardial infarction during follow-up, of which 8% were recorded only in primary care and 12% only in death registry records. A total of 959 470 people had a confirmed or suspected covid-19 diagnosis (714 162 in primary care data, 126 349 in hospital admission records, 776 503 in covid-19 laboratory test data, and 50 504 in death registry records). Although 58% of these were recorded in both primary care and covid-19 laboratory test data, 15% and 18%, respectively, were recorded in only one. Conclusions: This population-wide resource shows the importance of linking person level data across health settings to maximise completeness of key characteristics and to ascertain cardiovascular events and covid-19 diagnoses. Although this resource was initially established to support research on covid-19 and cardiovascular disease to benefit clinical care and public health and to inform healthcare policy, it can broaden further to enable a wide range of research

Characterising murine hippocampal iron homeostasis, in relation to markers of brain inflammation and metabolism, during ageing

Metal ions (Fe, Cu, and Zn) are essential to a healthy brain function, with the amount, localisation, and chemical form often tightly controlled. Evidence points towards loss of metal ion homeostasis within the ageing brain; in particular brain Fe accumulation appears to be a hallmark of ageing, which may place the brain at a greater risk of neurodegenerative disease. Unfortunately, the cause or consequence of altered brain metal ion homeostasis during ageing remains unknown, and there is a lack of data comparing brain metal ion homeostasis with other events of the ageing process (e.g. brain metabolism, brain inflammation). This study has utilised a multi-modal approach that incorporated: X-ray fluorescence microscopy for elemental mapping of metal ion homeostasis, Perl's Fe histochemistry, FTIR spectroscopic biochemical imaging of lactate and protein aggregates, and immuno-fluorescence analysis of markers of brain inflammation and Fe storage proteins (heavy-chain ferritin, light-chain ferritin, and mitochondrial ferritin). Interestingly, while age-related Fe accumulation was observed in corpus callosum white matter of murine (C56BL/6J) brain tissue (concomitant with elevated levels of markers of brain inflammation and altered metabolism), Fe content was not altered within the hippocampus (a decrease in total Zn within the mossy fibres was observed). Ultimately, the results of this study demonstrate an important association between elevated brain Fe and brain inflammation during natural ageing. This study also highlights that future research is required to image different chemical forms of Fe with respect to changes in brain metabolism and inflammation, as well as localising these changes to specific cell types

Revealing differences in the chemical form of zinc in brain tissue using K-edge X-ray absorption near-edge structure spectroscopy

Zinc is a prominent trace metal required for normal memory function. Memory loss and cognitive decline during natural ageing and neurodegenerative disease have been associated with altered brain-Zn homeostasis. Yet, the exact chemical pathways through which Zn influences memory function during health, natural ageing, or neurodegenerative disease remain unknown. The gap in the literature may in part be due to the difficulty to simultaneously image, and therefore, study the different chemical forms of Zn within the brain (or biological samples in general). To this extent, we have begun developing and optimising protocols that incorporate X-ray absorption near-edge structure (XANES) spectroscopic analysis of tissue at the Zn K-edge as an analytical tool to study Zn speciation in the brain. XANES is ideally suited for this task as all chemical forms of Zn are detected, the technique requires minimal sample preparation that may otherwise redistribute or alter the chemical form of Zn, and the Zn K-edge has known sensitivity to coordination geometry and ligand type. Herein, we report our initial results where we fit K-edge spectra collected from micro-dissected flash-frozen brain tissue, to a spectral library prepared from standard solutions, to demonstrate differences in the chemical form of Zn that exist between two brain regions, the hippocampus and cerebellum. Lastly, we have used an X-ray microprobe to demonstrate differences in Zn speciation within sub-regions of thin air-dried sections of the murine hippocampus; but, the corresponding results highlight that the chemical form of Zn is easily perturbed by sample preparation such as tissue sectioning or air-drying, which must be a critical consideration for future work