| 1. |
- Klionsky, Daniel J., et al.
(author)
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Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
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In: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
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Research review (peer-reviewed)abstract
- In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
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| 2. |
- Bèchet, Nicholas Burdon, et al.
(author)
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Glymphatic pathways in the gyrencephalic brain
- 2021
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In: Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism. - 1559-7016.
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Journal article (peer-reviewed)abstract
- Identification of the perivascular compartment as the point of exchange between cerebrospinal fluid (CSF) and interstitial fluid mediating solute clearance in the brain, named the glymphatic system, has emerged as an important clearance pathway for neurotoxic peptides such as amyloid-beta. However, the foundational science of the glymphatic system is based on rodent studies. Here we investigated whether the glymphatic system exists in a large mammal with a highly gyrified brain. CSF penetration into the brain via perivascular pathways, a hallmark of glymphatic function, was seen throughout the gyrencephalic cortex and subcortical structures, validating the conservation of the glymphatic system in a large mammal. Macroscopic CSF tracer distribution followed the sulci and fissures showing that these folds enhance CSF dispersion. Three-dimensional renditions from light sheet microscopy showed a PVS influx density 4-fold larger in the pig brain than in mice. This demonstrates the existence of an advanced solute transport system in the gyrencephalic brain that could be utilised therapeutically for enhancing waste clearance.
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| 3. |
- Bèchet, Nicholas B., et al.
(author)
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Light sheet fluorescence microscopy of optically cleared brains for studying the glymphatic system
- 2020
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In: Journal of Cerebral Blood Flow and Metabolism. - 0271-678X. ; 40:10, s. 1975-1986
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Journal article (peer-reviewed)abstract
- Fluid transport in the perivascular space by the glia-lymphatic (glymphatic) system is important for the removal of solutes from the brain parenchyma, including peptides such as amyloid-beta which are implicated in the pathogenesis of Alzheimer’s disease. The glymphatic system is highly active in the sleep state and under the influence of certain of anaesthetics, while it is suppressed in the awake state and by other anaesthetics. Here we investigated whether light sheet fluorescence microscopy of whole optically cleared murine brains was capable of detecting glymphatic differences in sleep- and awake-mimicking anaesthesia, respectively. Using light-sheet imaging of whole brains, we found anaesthetic-dependent cerebrospinal fluid (CSF) influx differences, including reduced tracer influx along tertiary branches of the middle cerebral artery and reduced influx along dorsal and anterior penetrating arterioles, in the awake-mimicking anaesthesia. This study establishes that light sheet microscopy of optically cleared brains is feasible for quantitative analyses and can provide images of the entire glymphatic system in whole brains.
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| 4. |
- Bechet, Nicholas
(author)
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Developing a Porcine Model to Study the Glymphatic System
- 2022
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Doctoral thesis (other academic/artistic)abstract
- The glymphatic system is a brain-wide solute clearance system that has developed in the brain to clear metabolic waste during sleep. This clearance is mediated by advective fluxes of cerebrospinal fluid (CSF) along perivascular spaces (PVS) and through the brain. The movement of CSF from the PVS and through the brain is dependent on the polarised expression of aquaporin-4 (AQP4) at astrocyte endfeet, that project to form the outer PVS boundary. The capacity of the glymphatic system to clear proteins like amyloid-beta (Aß) and tau has generated great interest in exploiting this system therapeutically in the context of Alzheimer’s disease (AD). However, much of the knowledge on the glymphatic system, more specifically, the microscopic machinery and processes, have been studied and described exclusively in rodents. To this end, apart from several magnetic resonance imaging studies confirming the existence of macroscopic glymphatic phenomena in humans i.e. advective movement of gadobutrol from the CSF into the brain, the microscopic aspects of the glymphatic system remain largely unexplored in the large gyrencephalic brain. Thus, the aims of this thesis were to develop a large animal model to study the glymphatic system and further use this model to study the glymphatic system in the context of neuropathology, which in this case took the form of sub-dural haematoma, and amyloid-beta (re)-circulation in the context of AD. To achieve this, a cisterna magna (CM) cannulation surgery was translated from rodents to pigs, such that it would be possible to introduce fluorescent tracers into the porcine CSF in vivo and explore the glymphatic pathways. Initially, to characterise these pathways whole brains were extracted intact after fluorescent tracer circulation and processed using several advanced imaging readouts, including macroscopy, along with confocal, light sheet and electron microscopy. Imaging data revealed: 1) The folded architecture of the gyrencephalic brain helped direct upstream CSF distribution, 2) PVS-mediated CSF influx into the brain is steadfast in the pig brain, as in rodents, and could be traced in deep sub-cortical structures and down to a capillary level, 3) PVS influx sites are 4-fold more extensive in pigs than in rodents. Taken together these data indicate not only a conservation of the glymphatic system and its machinery from rodents to pigs but a more developed system in the large mammal. In the context of suspected sub-acute subdural haematoma (SDH) a brain-wide impairment in glymphatic influx amounted, raising important questions concerning the consequence of undiagnosed SDH for glymphatic function and brain clearance. In the context of AD the acute introduction of Aß1-42 into the CSF was found to impair glymphatic function, highlighting the consequences of Aß recirculation for brain clearance. Interestingly, upon closer examination it was found that Aß did not penetrate into the brain as was the case with inert protein tracers, but instead remained localised to pial and penetrating arteries. This localisation was elastin specific and only occured with Aß (1-40/1-42) but not dextran or bovine serum albumin. This outcome appears to reflect an Aß entrapment system that prevents the recirculation of Aß into the brain, but further work is necessary to unravel its potential as a clearance pathway for Aß from the CSF to protein transporters at the endothelial cell layer. In an attempt to study this in vivo a porcine cranial window model was generated in order to image PVS transport in the large gyrencephalic brain. While low resolution imaging was indeed possible, brain motion proved a challenge not yet overcome. We hope that in the future, through mitigating brain motion, it will be possible to study PVS transport, the glymphatic system, and this potential Aß entrapment pathway, in the brain of a large mammal in vivo.
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| 5. |
- Bechet, Nicholas, et al.
(author)
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Direct Cannula Implantation in the Cisterna Magna of Pigs
- 2021
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In: Journal of Visualized Experiments. - : MyJove Corporation. - 1940-087X. ; 172, s. 1-12
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Journal article (peer-reviewed)abstract
- The glymphatic system is a waste clearance system in the brain that relies on the flow of cerebrospinal fluid (CSF) in astrocyte-bound perivascular spaces and has been implicated in the clearance of neurotoxic peptides such as amyloid-beta. Impaired glymphatic function exacerbates disease pathology in animal models of neurodegenerative diseases, such as Alzheimer's, which highlights the importance of understanding this clearance system. The glymphatic system is often studied by cisterna magna cannulations (CMc), where tracers are delivered directly into the cerebrospinal fluid (CSF). Most studies, however, have been carried out in rodents. Here, we demonstrate an adaptation of the CMc technique in pigs. Using CMc in pigs, the glymphatic system can be studied at a high optical resolution in gyrencephalic brains and in doing so bridges the knowledge gap between rodent and human glymphatics.
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| 6. |
- Lindstedt, Sandra, et al.
(author)
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High resolution fluorescence imaging of the alveolar scaffold as a novel tool to assess lung injury
- 2024
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In: Scientific Reports. - 2045-2322. ; 14:1
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Journal article (peer-reviewed)abstract
- Acute lung injury (ALI) represents an aetiologically diverse form of pulmonary damage. Part of the assessment and diagnosis of ALI depends on skilled observer-based scoring of brightfield microscopy tissue sections. Although this readout is sufficient to determine gross alterations in tissue structure, its categorical scores lack the sensitivity to describe more subtle changes in lung morphology. To generate a more sensitive readout of alveolar perturbation we carried out high resolution immunofluorescence imaging on 200 μm lung vibratome sections from baseline and acutely injured porcine lung tissue, stained with a tomato lectin, Lycopersicon Esculentum Dylight-488. With the ability to resolve individual alveoli along with their inner and outer wall we generated continuous readouts of alveolar wall thickness and circularity. From 212 alveoli traced from 10 baseline lung samples we established normal distributions for alveolar wall thickness (27.37; 95% CI [26.48:28.26]) and circularity (0.8609; 95% CI [0.8482:0.8667]) in healthy tissue. Compared to acutely injured lung tissue baseline tissue exhibited a significantly lower wall thickness (26.86 ± 0.4998 vs 50.55 ± 4.468; p = 0.0003) and higher degree of circularityϕ≤ (0.8783 ± 0.01965 vs 0.4133 ± 0.04366; p < 0.0001). These two components were subsequently combined into a single more sensitive variable, termed the morphological quotient (MQ), which exhibited a significant negative correlation (R 2 = 0.9919, p < 0.0001) with the gold standard of observer-based scoring. Through the utilisation of advanced light imaging we show it is possible to generate sensitive continuous datasets describing fundamental morphological changes that arise in acute lung injury. These data represent valuable new analytical tools that can be used to precisely benchmark changes in alveolar morphology both in disease/injury as well as in response to treatment/therapy.
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| 7. |
- Mittendorfer, Margareta, et al.
(author)
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Restoring discarded porcine lungs by ex vivo removal of neutrophil extracellular traps
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In: The Journal of heart and lung transplantation : the official publication of the International Society for Heart Transplantation. - 1557-3117.
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Journal article (peer-reviewed)abstract
- BACKGROUND: By causing inflammation and tissue damage, neutrophil extracellular traps (NETs) constitute an underlying mechanism of aspiration-induced lung injury, a major factor of the low utilization of donor lungs in lung transplantation (LTx).METHOD: To determine whether NET removal during ex vivo lung perfusion (EVLP) can restore lung function and morphology in aspiration-damaged lungs, gastric aspiration lung injury was induced in 12 pigs. After confirmation of acute respiratory distress syndrome, the lungs were explanted and assigned to NET removal connected to EVLP (treated) (n=6) or EVLP only (non-treated) (n=6). Hemodynamic measurements were taken, and blood and tissue samples were collected to assess lung function, morphology, levels of cell-free DNA, extracellular histones, and nucleosomes as markers of NETs as well as cytokine levels.RESULTS: After EVLP and NET removal in porcine lungs, PaO 2/FiO 2 ratios increased significantly compared to those undergoing EVLP alone (p=0.0411). Treated lungs had lower cell-free DNA (p=0.0260) and lower levels of extracellular histones in EVLP perfusate than non-treated lungs (p=0.0260). According to histopathology, treated lungs showed less immune cell infiltration and less oedema compared with non-treated lungs which was reflected in decreased levels of pro-inflammatory cytokines in EVLP perfusate and BALF. CONCLUSION: To conclude, removing NETs during EVLP improved lung function and morphology in aspiration-damaged donor lungs. The ability to remove NETs during EVLP could represent a new therapeutic approach for LTx and potentially expand the donor pool for transplantation.
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| 8. |
- Munk, Anne Sofie, et al.
(author)
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PDGF-B Is Required for Development of the Glymphatic System
- 2019
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In: Cell Reports. - : Elsevier BV. - 2211-1247. ; , s. 3-2969
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Journal article (peer-reviewed)abstract
- The glymphatic system is a highly polarized cerebrospinal fluid (CSF) transport system that facilitates the clearance of neurotoxic molecules through a brain-wide network of perivascular pathways. Herein we have mapped the development of the glymphatic system in mice. Perivascular CSF transport first emerges in hippocampus in newborn mice, and a mature glymphatic system is established in the cortex at 2 weeks of age. Formation of astrocytic endfeet and polarized expression of aquaporin 4 (AQP4) consistently coincided with the appearance of perivascular CSF transport. Deficiency of platelet-derived growth factor B (PDGF-B) function in the PDGF retention motif knockout mouse line Pdgfb ret/ret suppressed the development of the glymphatic system, whose functions remained suppressed in adulthood compared with wild-type mice. These experiments map the natural development of the glymphatic system in mice and define a critical role of PDGF-B in the development of perivascular CSF transport.
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| 9. |
- Niroomand, Anna, et al.
(author)
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Proteomic Analysis of Primary Graft Dysfunction in Porcine Lung Transplantation Reveals Alveolar-Capillary Barrier Changes Underlying the High Particle Flow Rate in Exhaled Breath
- 2024
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In: Transplant International. - 0934-0874. ; 37
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Journal article (peer-reviewed)abstract
- Primary graft dysfunction (PGD) remains a challenge for lung transplantation (LTx) recipients as a leading cause of poor early outcomes. New methods are needed for more detailed monitoring and understanding of the pathophysiology of PGD. The measurement of particle flow rate (PFR) in exhaled breath is a novel tool to monitor and understand the disease at the proteomic level. In total, 22 recipient pigs underwent orthotopic left LTx and were evaluated for PGD on postoperative day 3. Exhaled breath particles (EBPs) were evaluated by mass spectrometry and the proteome was compared to tissue biopsies and bronchoalveolar lavage fluid (BALF). Findings were confirmed in EBPs from 11 human transplant recipients. Recipients with PGD had significantly higher PFR [686.4 (449.7–8,824.0) particles per minute (ppm)] compared to recipients without PGD [116.6 (79.7–307.4) ppm, p = 0.0005]. Porcine and human EBP proteins recapitulated proteins found in the BAL, demonstrating its utility instead of more invasive techniques. Furthermore, adherens and tight junction proteins were underexpressed in PGD tissue. Histological and proteomic analysis found significant changes to the alveolar-capillary barrier explaining the high PFR in PGD. Exhaled breath measurement is proposed as a rapid and non-invasive bedside measurement of PGD.
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| 10. |
- Ramos, Marta, et al.
(author)
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Cisterna Magna Injection in Rats to Study Glymphatic Function
- 2019
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In: Methods in molecular biology (Clifton, N.J.). - New York, NY : Springer New York. - 1940-6029. ; 1938, s. 97-104
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Journal article (peer-reviewed)abstract
- The recently discovered glymphatic system, which supports brain-wide clearance of metabolic waste, has become the subject of intense research within the past few years. Its nomenclature arose due to its functionally analogous nature to the lymphatic system in combination with glial cells that are part of its anatomical boundaries. The influx of cerebrospinal fluid (CSF) from perivascular spaces into the brain interstitium acts to clear intraparenchymal solutes. CSF is produced by the choroid plexus and flows from the ventricles to the subarachnoid space via the cisterna magna, and as such the injection of tracer molecules into any one of these spaces could be used for studying CSF movement through the glymphatic system. Of these options, the cisterna magna is most favorable as it offers a route of entry that does not involve craniotomy. Herein we describe the cisterna magna (CM) injection procedure carried out in rats, essential for studying glymphatic influx and efflux dynamics.
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