Point of Care Ultrasound Bladder Volume Calculation on the Acute Medical Unit.

Nursing staff, healthcare assistants (HCAs) and other healthcare professionals on the Acute Medical Unit (AMU) at Royal Berkshire Hospital (RBH) were taught a Point of Care Ultrasound (POCUS) skill during a twenty minute session. Practitioners learned how to take bladder volume measurements with the Butterfly iQ, a portable ultrasound device which provides a visually-aided method of volume measurement. A Likert scale was used to measure the confidence that staff had in performing volume measurements with the AMU automated scanners, and with the semi-automated Butterfly iQ. After the teaching session, confidence reported by practitioners in using the semi-automated visual method was significantly higher than confidence reported in using the automated non-visual scanners (t < 0.001). Minimal time and expense was required to teach practitioners how to perform this skill. Training nurses in POCUS for bladder visualisation and bladder volume calculation is easy and practicable.

The endotoxin hypothesis of Alzheimer's disease.

Lipopolysaccharide (LPS) constitutes much of the surface of Gram-negative bacteria, and if LPS enters the human body or brain can induce inflammation and act as an endotoxin. We outline the hypothesis here that LPS may contribute to the pathophysiology of Alzheimer's disease (AD) via peripheral infections or gut dysfunction elevating LPS levels in blood and brain, which promotes: amyloid pathology, tau pathology and microglial activation, contributing to the neurodegeneration of AD. The evidence supporting this hypothesis includes: i) blood and brain levels of LPS are elevated in AD patients, ii) AD risk factors increase LPS levels or response, iii) LPS induces Aß expression, aggregation, inflammation and neurotoxicity, iv) LPS induces TAU phosphorylation, aggregation and spreading, v) LPS induces microglial priming, activation and neurotoxicity, and vi) blood LPS induces loss of synapses, neurons and memory in AD mouse models, and cognitive dysfunction in humans. However, to test the hypothesis, it is necessary to test whether reducing blood LPS reduces AD risk or progression. If the LPS endotoxin hypothesis is correct, then treatments might include: reducing infections, changing gut microbiome, reducing leaky gut, decreasing blood LPS, or blocking LPS response.

Disease phenotypic screening in neuron-glia cocultures identifies blockers of inflammatory neurodegeneration.

Neuropathology is often mediated by interactions between neurons and glia that cannot be modeled by monocultures. However, cocultures are difficult to use and analyze for high-content screening. Here, we perform compound screening using primary neuron-glia cultures to model inflammatory neurodegeneration, live-cell stains, and automated classification of neurons, astrocytes or microglia using open-source software. Out of 227 compounds with known bioactivities, 29 protected against lipopolysaccharide-induced neuronal loss, including drugs affecting adrenergic, steroid, inflammatory and MAP kinase signaling. The screen also identified physiological compounds, such as noradrenaline and progesterone, that protected and identified neurotoxic compounds, such as a TLR7 agonist, that induced microglial proliferation. Most compounds used here have not been tested in a neuron-glia coculture neurodegeneration assay previously. Thus, combining a complex cellular disease model with high-content screening of known compounds and automated image analysis allows identification of important biology, as well as potential targets and drugs for treatment.

Cell death by phagocytosis.

Cells can die as a consequence of being phagocytosed by other cells - a form of cell death that has been called phagotrophy, cell cannibalism, programmed cell removal and primary phagocytosis. However, these are all different manifestations of cell death by phagocytosis (termed 'phagoptosis' for short). The engulfed cells die as a result of cytotoxic oxidants, peptides and degradative enzymes within acidic phagolysosomes. Cell death by phagocytosis was discovered by Metchnikov in the 1880s, but was neglected until recently. It is now known to contribute to developmental cell death in nematodes, Drosophila and mammals, and is central to innate and adaptive immunity against pathogens. Cell death by phagocytosis mediates physiological turnover of erythrocytes and other leucocytes, making it the most abundant form of cell death in the mammalian body. Immunity against cancer is also partly mediated by macrophage phagocytosis of cancer cells, but cancer cells can also phagocytose host cells and other cancer cells in order to survive. Recent evidence indicates neurodegeneration and other neuropathologies can be mediated by microglial phagocytosis of stressed neurons. Thus, despite cell death by phagocytosis being poorly recognized, it is one of the oldest, commonest and most important forms of cell death.

LRPAP1 is released from activated microglia and inhibits microglial phagocytosis and amyloid beta aggregation.

Low-density lipoprotein receptor-related protein-associated protein 1 (LRPAP1), also known as receptor associated protein (RAP), is an endoplasmic reticulum (ER) chaperone and inhibitor of LDL receptor related protein 1 (LRP1) and related receptors. These receptors have dozens of physiological ligands and cell functions, but it is not known whether cells release LRPAP1 physiologically at levels that regulate these receptors and cell functions. We used mouse BV-2 and human CHME3 microglial cell lines, and found that microglia released nanomolar levels of LRPAP1 when inflammatory activated by lipopolysaccharide or when ER stressed by tunicamycin. LRPAP1 was found on the surface of live activated and non-activated microglia, and anti-LRPAP1 antibodies induced internalization. Addition of 10 nM LRPAP1 inhibited microglial phagocytosis of isolated synapses and cells, and the uptake of Aß. LRPAP1 also inhibited Aß aggregation in vitro. Thus, activated and stressed microglia release LRPAP1 levels that can inhibit phagocytosis, Aß uptake and Aß aggregation. We conclude that LRPAP1 release may regulate microglial functions and Aß pathology, and more generally that extracellular LRPAP1 may be a physiological and pathological regulator of a wide range of cell functions.

P2Y6 Receptor-Dependent Microglial Phagocytosis of Synapses during Development Regulates Synapse Density and Memory.

During brain development, excess synapses are pruned (i.e., removed), in part by microglial phagocytosis, and dysregulated synaptic pruning can lead to behavioral deficits. The P2Y6 receptor (P2Y6R) is known to regulate microglial phagocytosis of neurons, and to regulate microglial phagocytosis of synapses in cell culture and in vivo during aging. However, currently it is unknown whether P2Y6R regulates synaptic pruning during development. Here, we show that P2Y6R KO mice of both sexes had strongly reduced microglial internalization of synaptic material, measured as Vglut1 within CD68-staining lysosomes of microglia at postnatal day 30 (P30), suggesting reduced microglial phagocytosis of synapses. Consistent with this, we found an increased density of synapses in the somatosensory cortex and the CA3 region and dentate gyrus of the hippocampus at P30. We also show that adult P2Y6R KO mice have impaired short- and long-term spatial memory and impaired short- and long-term recognition memory compared with WT mice, as measured by novel location recognition, novel object recognition, and Y-maze memory tests. Overall, this indicates that P2Y6R regulates microglial phagocytosis of synapses during development, and this contributes to memory capacity.SIGNIFICANCE STATEMENT The P2Y6 receptor (P2Y6R) is activated by uridine diphosphate released by neurons, inducing microglial phagocytosis of such neurons or synapses. We tested whether P2Y6R regulates developmental synaptic pruning in mice and found that P2Y6R KO mice have reduced synaptic material within microglial lysosomes, and increased synaptic density in the brains of postnatal day 30 mice, consistent with reduced synaptic pruning during development. We also found that adult P2Y6R KO mice had reduced memory, consistent with persistent deficits in brain function, resulting from impaired synaptic pruning. Overall, the results suggest that P2Y6R mediates microglial phagocytosis of synapses during development, and the absence of this results in memory deficits in the adult.

Extracellular tau stimulates phagocytosis of living neurons by activated microglia via Toll-like 4 receptor-NLRP3 inflammasome-caspase-1 signalling axis.

In tauopathies, abnormal deposition of intracellular tau protein followed by gradual elevation of tau in cerebrospinal fluids and neuronal loss has been documented, however, the mechanism how actually neurons die under tau pathology is largely unknown. We have previously shown that extracellular tau protein (2N4R isoform) can stimulate microglia to phagocytose live neurons, i.e. cause neuronal death by primary phagocytosis, also known as phagoptosis. Here we show that tau protein induced caspase-1 activation in microglial cells via 'Toll-like' 4 (TLR4) receptors and neutral sphingomyelinase. Tau-induced neuronal loss was blocked by caspase-1 inhibitors (Ac-YVAD-CHO and VX-765) as well as by TLR4 antibodies. Inhibition of caspase-1 by Ac-YVAD-CHO prevented tau-induced exposure of phosphatidylserine on the outer leaflet of neuronal membranes and reduced microglial phagocytic activity. We also show that suppression of NLRP3 inflammasome, which is down-stream of TLR4 receptors and mediates caspase-1 activation, by a specific inhibitor (MCC550) also prevented tau-induced neuronal loss. Moreover, NADPH oxidase is also involved in tau-induced neurotoxicity since neuronal loss was abolished by its pharmacological inhibitor. Overall, our data indicate that extracellular tau protein stimulates microglia to phagocytose live neurons via Toll-like 4 receptor-NLRP3 inflammasome-caspase-1 axis and NADPH oxidase, each of which may serve as a potential molecular target for pharmacological treatment of tauopathies.

The Endotoxin Hypothesis of Parkinson's Disease.

The endotoxin hypothesis of Parkinson's disease (PD) is the idea that lipopolysaccharide (LPS) endotoxins contribute to the pathogenesis of this disorder. LPS endotoxins are found in, and released from, the outer membrane of Gram-negative bacteria, for example in the gut. It is proposed that gut dysfunction in early PD leads to elevated LPS levels in the gut wall and blood, which promotes both α-synuclein aggregation in the enteric neurons and a peripheral inflammatory response. Communication to the brain via circulating LPS and cytokines in the blood and/or the gut-brain axis leads to neuroinflammation and spreading of α-synuclein pathology, exacerbating neurodegeneration in brainstem nuclei and loss of dopaminergic neurons in the substantia nigra, and manifesting in the clinical symptoms of PD. The evidence supporting this hypothesis includes: (1) gut dysfunction, permeability, and bacterial changes occur early in PD, (2) serum levels of LPS are increased in a proportion of PD patients, (3) LPS induces α-synuclein expression, aggregation, and neurotoxicity, (4) LPS causes activation of peripheral monocytes leading to inflammatory cytokine production, and (5) blood LPS causes brain inflammation and specific loss of midbrain dopaminergic neurons, mediated by microglia. If the hypothesis is correct, then treatment options might include: (1) changing the gut microbiome, (2) reducing gut permeability, (3) reducing circulating LPS levels, or (4) blocking the response of immune cells and microglia to LPS. However, the hypothesis has a number of limitations and requires further testing, in particular whether reducing LPS levels can reduce PD incidence, progression, or severity. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Syk inhibitors protect against microglia-mediated neuronal loss in culture.

Microglia are brain macrophages and play beneficial and/or detrimental roles in many brain pathologies because of their inflammatory and phagocytic activity. Microglial inflammation and phagocytosis are thought to be regulated by spleen tyrosine kinase (Syk), which is activated by multiple microglial receptors, including TREM2 (Triggering Receptor Expressed on Myeloid Cells 2), implicated in neurodegeneration. Here, we have tested whether Syk inhibitors can prevent microglia-dependent neurodegeneration induced by lipopolysaccharide (LPS) in primary neuron-glia cultures. We found that the Syk inhibitors BAY61-3606 and P505-15 (at 1 and 10 µM, respectively) completely prevented the neuronal loss induced by LPS, which was microglia-dependent. Syk inhibition also prevented the spontaneous loss of neurons from older neuron-glia cultures. In the absence of LPS, Syk inhibition depleted microglia from the cultures and induced some microglial death. However, in the presence of LPS, Syk inhibition had relatively little effect on microglial density (reduced by 0-30%) and opposing effects on the release of two pro-inflammatory cytokines (IL-6 decreased by about 45%, TNFα increased by 80%). Syk inhibition also had no effect on the morphological transition of microglia exposed to LPS. On the other hand, inhibition of Syk reduced microglial phagocytosis of beads, synapses and neurons. Thus, Syk inhibition in this model is most likely neuroprotective by reducing microglial phagocytosis, however, the reduced microglial density and IL-6 release may also contribute. This work adds to increasing evidence that Syk is a key regulator of the microglial contribution to neurodegenerative disease and suggests that Syk inhibitors may be used to prevent excessive microglial phagocytosis of synapses and neurons.

Alzheimer's disease-associated R47H TREM2 increases, but wild-type TREM2 decreases, microglial phagocytosis of synaptosomes and neuronal loss.

Triggering receptor on myeloid cells 2 (TREM2) is an innate immune receptor, upregulated on the surface of microglia associated with amyloid plaques in Alzheimer's disease (AD). Individuals heterozygous for the R47H variant of TREM2 have greatly increased risk of developing AD. We examined the effects of wild-type (WT), R47H and knock-out (KO) of human TREM2 expression in three microglial cell systems. Addition of mouse BV-2 microglia expressing R47H TREM2 to primary mouse neuronal cultures caused neuronal loss, not observed with WT TREM2. Neuronal loss was prevented by using annexin V to block exposed phosphatidylserine, an eat-me signal and ligand of TREM2, suggesting loss was mediated by microglial phagocytosis of neurons exposing phosphatidylserine. Addition of human CHME-3 microglia expressing R47H TREM2 to LUHMES neuronal-like cells also caused loss compared to WT TREM2. Expression of R47H TREM2 in BV-2 and CHME-3 microglia increased their uptake of phosphatidylserine-beads and synaptosomes versus WT TREM2. Human iPSC-derived microglia with heterozygous R47H TREM2 had increased phagocytosis of synaptosomes vs common-variant TREM2. Additionally, phosphatidylserine liposomes increased activation of human iPSC-derived microglia expressing homozygous R47H TREM2 versus common-variant TREM2. Finally, overexpression of TREM2 in CHME-3 microglia caused increased expression of cystatin F, a cysteine protease inhibitor, and knock-down of cystatin F increased CHME-3 uptake of phosphatidylserine-beads. Together, these data suggest that R47H TREM2 may increase AD risk by increasing phagocytosis of synapses and neurons via greater activation by phosphatidylserine and that WT TREM2 may decrease microglial phagocytosis of synapses and neurons via cystatin F.

P2Y6 receptor-dependent microglial phagocytosis of synapses mediates synaptic and memory loss in aging.

Aging causes loss of brain synapses and memory, and microglial phagocytosis of synapses may contribute to this loss. Stressed neurons can release the nucleotide UTP, which is rapidly converted into UDP, that in turn activates the P2Y6 receptor (P2Y6 R) on the surface of microglia, inducing microglial phagocytosis of neurons. However, whether the activation of P2Y6 R affects microglial phagocytosis of synapses is unknown. We show here that inactivation of P2Y6 R decreases microglial phagocytosis of isolated synapses (synaptosomes) and synaptic loss in neuronal-glial co-cultures. In vivo, wild-type mice aged from 4 to 17 months exhibited reduced synaptic density in cortical and hippocampal regions, which correlated with increased internalization of synaptic material within microglia. However, this aging-induced synaptic loss and internalization were absent in P2Y6 R knockout mice, and these mice also lacked any aging-induced memory loss. Thus, P2Y6 R appears to mediate aging-induced loss of synapses and memory by increasing microglial phagocytosis of synapses. Consequently, blocking P2Y6 R has the potential to prevent age-associated memory impairment.

Activated microglia release ß-galactosidase that promotes inflammatory neurodegeneration.

Beta (ß)-galactosidase is a lysosomal enzyme that removes terminal galactose residues from glycolipids and glycoproteins. It is upregulated in, and used as a marker for, senescent cells. Microglia are brain macrophages implicated in neurodegeneration, and can upregulate ß-galactosidase when senescent. We find that inflammatory activation of microglia induced by lipopolysaccharide results in translocation of ß-galactosidase to the cell surface and release into the medium. Similarly, microglia in aged mouse brains appear to have more ß-galactosidase on their surface. Addition of ß-galactosidase to neuronal-glial cultures causes microglial activation and neuronal loss mediated by microglia. Inhibition of ß-galactosidase in neuronal-glial cultures reduces inflammation and neuronal loss induced by lipopolysaccharide. Thus, activated microglia release ß-galactosidase that promotes microglial-mediated neurodegeneration which is prevented by inhibition of ß-galactosidase.

Microglia states and nomenclature: A field at its crossroads.

Microglial research has advanced considerably in recent decades yet has been constrained by a rolling series of dichotomies such as "resting versus activated" and "M1 versus M2." This dualistic classification of good or bad microglia is inconsistent with the wide repertoire of microglial states and functions in development, plasticity, aging, and diseases that were elucidated in recent years. New designations continuously arising in an attempt to describe the different microglial states, notably defined using transcriptomics and proteomics, may easily lead to a misleading, although unintentional, coupling of categories and functions. To address these issues, we assembled a group of multidisciplinary experts to discuss our current understanding of microglial states as a dynamic concept and the importance of addressing microglial function. Here, we provide a conceptual framework and recommendations on the use of microglial nomenclature for researchers, reviewers, and editors, which will serve as the foundations for a future white paper.

Neu1 Is Released From Activated Microglia, Stimulating Microglial Phagocytosis and Sensitizing Neurons to Glutamate.

Neuraminidase 1 (Neu1) hydrolyses terminal sialic acid residues from glycoproteins and glycolipids, and is normally located in lysosomes, but can be released onto the surface of activated myeloid cells and microglia. We report that endotoxin/lipopolysaccharide-activated microglia released Neu1 into culture medium, and knockdown of Neu1 in microglia reduced both Neu1 protein and neuraminidase activity in the culture medium. Release of Neu1 was reduced by inhibitors of lysosomal exocytosis, and accompanied by other lysosomal proteins, including protective protein/cathepsin A, known to keep Neu1 active. Extracellular neuraminidase or over-expression of Neu1 increased microglial phagocytosis, while knockdown of Neu1 decreased phagocytosis. Microglial activation caused desialylation of microglial phagocytic receptors Trem2 and MerTK, and increased binding to Trem2 ligand galectin-3. Culture media from activated microglia contained Neu1, and when incubated with neurons induced their desialylation, and increased the neuronal death induced by low levels of glutamate. Direct desialylation of neurons by adding sialidase or inhibiting sialyltransferases also increased glutamate-induced neuronal death. We conclude that activated microglia can release active Neu1, possibly by lysosomal exocytosis, and this can both increase microglial phagocytosis and sensitize neurons to glutamate, thus potentiating neuronal death.

Brain Cells Release Calreticulin That Attracts and Activates Microglia, and Inhibits Amyloid Beta Aggregation and Neurotoxicity.

Calreticulin is a chaperone, normally found in the endoplasmic reticulum, but can be released by macrophages into the extracellular medium. It is also found in cerebrospinal fluid bound to amyloid beta (Aß). We investigated whether brain cells release calreticulin, and whether extracellular calreticulin had any effects on microglia and neurons relevant to neuroinflammation and neurodegeneration. We found that microglia release nanomolar levels of calreticulin when inflammatory-activated with lipopolysaccharide, when endoplasmic reticulum stress was induced by tunicamycin, or when cell death was induced by staurosporine, and that neurons release calreticulin when crushed. Addition of nanomolar levels of extracellular calreticulin was found to chemoattract microglia, and activate microglia to release cytokines TNF-α, IL-6 and IL-1ß, as well as chemokine (C-C motif) ligand 2. Calreticulin blocked Aß fibrillization and modified Aß oligomerization, as measured by thioflavin T fluorescence and transmission electron microscopy. Extracellular calreticulin also altered microglial morphology and proliferation, and prevented Aß-induced neuronal loss in primary neuron-glial cultures. Thus, calreticulin is released by microglia and neurons, and acts: as an alarmin to recruit and activate microglia, as an extracellular chaperone to prevent Aß aggregation, and as a neuroprotectant against Aß neurotoxicity.

Acoustic Screening for Obstructive Sleep Apnea in Home Environments Based on Deep Neural Networks.

Obstructive sleep apnea (OSA) is a chronic and prevalent condition with well-established comorbidities. However, many severe cases remain undiagnosed due to poor access to polysomnography (PSG), the gold standard for Obstructive sleep apnea (OSA) diagnosis. Accurate home-based methods to screen for OSA are needed, which can be applied inexpensively to high-risk subjects to identify those that require PSG to fully assess their condition. A number of methods that analyse speech or breathing sounds to screen for OSA have been previously investigated. However, these methods have constraints that limit their use in home environments (e.g., they require specialised equipment, are not robust to background noise, are obtrusive or depend on tightly controlled conditions). This paper proposes a novel method to screen for OSA, which analyses sleep breathing sounds recorded with a smartphone at home. Audio recordings made over a whole night are divided into segments, each of which is classified for the presence or absence of OSA by a deep neural network. The apnea-hypopnea index estimated from the segments predicted as containing evidence of OSA is then used to screen for the condition. Audio recordings made during home sleep apnea testing from 103 participants for 1 or 2 nights were used to develop and evaluate the proposed system. When screening for moderate OSA the acoustics based system achieved a sensitivity of 0.79 and a specificity of 0.80. The sensitivity and specificity when screening for severe OSA were 0.78 and 0.93, respectively. The system is suitable for implementation on consumer smartphones.

Knockout of the P2Y6 Receptor Prevents Peri-Infarct Neuronal Loss after Transient, Focal Ischemia in Mouse Brain.

After stroke, there is a delayed neuronal loss in brain areas surrounding the infarct, which may in part be mediated by microglial phagocytosis of stressed neurons. Microglial phagocytosis of stressed or damaged neurons can be mediated by UDP released from stressed neurons activating the P2Y6 receptor on microglia, inducing microglial phagocytosis of such neurons. We show evidence here from a small trial that the knockout of the P2Y6 receptor, required for microglial phagocytosis of neurons, prevents the delayed neuronal loss after transient, focal brain ischemia induced by endothelin-1 injection in mice. Wild-type mice had neuronal loss and neuronal nuclear material within microglia in peri-infarct areas. P2Y6 receptor knockout mice had no significant neuronal loss in peri-infarct brain areas seven days after brain ischemia. Thus, delayed neuronal loss after stroke may in part be mediated by microglial phagocytosis of stressed neurons, and the P2Y6 receptor is a potential treatment target to prevent peri-infarct neuronal loss.

The microglial P2Y6 receptor mediates neuronal loss and memory deficits in neurodegeneration.

Microglia are implicated in neurodegeneration, potentially by phagocytosing neurons, but it is unclear how to block the detrimental effects of microglia while preserving their beneficial roles. The microglial P2Y6 receptor (P2Y6R) - activated by extracellular UDP released by stressed neurons - is required for microglial phagocytosis of neurons. We show here that injection of amyloid beta (Aß) into mouse brain induces microglial phagocytosis of neurons, followed by neuronal and memory loss, and this is all prevented by knockout of P2Y6R. In a chronic tau model of neurodegeneration (P301S TAU mice), P2Y6R knockout prevented TAU-induced neuronal and memory loss. In vitro, P2Y6R knockout blocked microglial phagocytosis of live but not dead targets and reduced tau-, Aß-, and UDP-induced neuronal loss in glial-neuronal cultures. Thus, the P2Y6 receptor appears to mediate Aß- and tau-induced neuronal and memory loss via microglial phagocytosis of neurons, suggesting that blocking this receptor may be beneficial in the treatment of neurodegenerative diseases.