Adenosine signalling to astrocytes coordinates brain metabolism and function.

Brain computation performed by billions of nerve cells relies on a sufficient and uninterrupted nutrient and oxygen supply1,2. Astrocytes, the ubiquitous glial neighbours of neurons, govern brain glucose uptake and metabolism3,4, but the exact mechanisms of metabolic coupling between neurons and astrocytes that ensure on-demand support of neuronal energy needs are not fully understood5,6. Here we show, using experimental in vitro and in vivo animal models, that neuronal activity-dependent metabolic activation of astrocytes is mediated by neuromodulator adenosine acting on astrocytic A2B receptors. Stimulation of A2B receptors recruits the canonical cyclic adenosine 3',5'-monophosphate-protein kinase A signalling pathway, leading to rapid activation of astrocyte glucose metabolism and the release of lactate, which supplements the extracellular pool of readily available energy substrates. Experimental mouse models involving conditional deletion of the gene encoding A2B receptors in astrocytes showed that adenosine-mediated metabolic signalling is essential for maintaining synaptic function, especially under conditions of high energy demand or reduced energy supply. Knockdown of A2B receptor expression in astrocytes led to a major reprogramming of brain energy metabolism, prevented synaptic plasticity in the hippocampus, severely impaired recognition memory and disrupted sleep. These data identify the adenosine A2B receptor as an astrocytic sensor of neuronal activity and show that cAMP signalling in astrocytes tunes brain energy metabolism to support its fundamental functions such as sleep and memory.

Discontinuation vs. continuation of renin-angiotensin system inhibition before non-cardiac surgery: the SPACE trial.

BACKGROUND AND AIMS: Haemodynamic instability is associated with peri-operative myocardial injury, particularly in patients receiving renin-angiotensin system (RAS) inhibitors (angiotensin-converting-enzyme inhibitors/angiotensin II receptor blockers). Whether stopping RAS inhibitors to minimise hypotension, or continuing RAS inhibitors to avoid hypertension, reduces peri-operative myocardial injury remains unclear. METHODS: From 31 July 2017 to 1 October 2021, patients aged ≥60 years undergoing elective non-cardiac surgery were randomly assigned to either discontinue or continue RAS inhibitors prescribed for existing medical conditions in six UK centres. Renin-angiotensin system inhibitors were withheld for different durations (2-3 days) before surgery, according to their pharmacokinetic profile. The primary outcome, masked to investigators, clinicians, and patients, was myocardial injury [plasma high-sensitivity troponin-T (hs-TnT) ≥ 15 ng/L within 48 h after surgery, or ≥5 ng/L increase when pre-operative hs-TnT ≥15 ng/L]. Pre-specified adverse haemodynamic events occurring within 48 h of surgery included acute hypertension (>180 mmHg) and hypotension requiring vasoactive therapy. RESULTS: Two hundred and sixty-two participants were randomized to continue (n = 132) or stop (n = 130) RAS inhibitors. Myocardial injury occurred in 58 (48.3%) patients randomized to discontinue, compared with 50 (41.3%) patients who continued, RAS inhibitors [odds ratio (for continuing): 0.77; 95% confidence interval (CI) 0.45-1.31]. Hypertensive adverse events were more frequent when RAS inhibitors were stopped [16 (12.4%)], compared with 7 (5.3%) who continued RAS inhibitors [odds ratio (for continuing): 0.4; 95% CI 0.16-1.00]. Hypotension rates were similar when RAS inhibitors were stopped [12 (9.3%)] or continued [11 (8.4%)]. CONCLUSIONS: Discontinuing RAS inhibitors before non-cardiac surgery did not reduce myocardial injury, and could increase the risk of clinically significant acute hypertension. These findings require confirmation in future studies.

Transcriptional response of the heart to vagus nerve stimulation.

Heart failure is a major clinical problem, with treatments involving medication, devices, and emerging neuromodulation therapies such as vagus nerve stimulation (VNS). Considering the ongoing interest in using VNS to treat cardiovascular disease, it is important to understand the genetic and molecular changes developing in the heart in response to this form of autonomic neuromodulation. This experimental animal (rat) study investigated the immediate transcriptional response of the ventricular myocardium to selective stimulation of vagal efferent activity using an optogenetic approach. Vagal preganglionic neurons in the dorsal motor nucleus of the vagus nerve were genetically targeted to express light-sensitive chimeric channelrhodopsin variant ChIEF and stimulated using light. RNA sequencing of the left ventricular myocardium identified 294 differentially expressed genes (false discovery rate < 0.05). Qiagen Ingenuity Pathway Analysis (IPA) highlighted 118 canonical pathways that were significantly modulated by vagal activity, of which 14 had a z score of ≥2/≤-2, including EIF-2, IL-2, integrin, and NFAT-regulated cardiac hypertrophy. IPA revealed the effect of efferent vagus stimulation on protein synthesis, autophagy, fibrosis, autonomic signaling, inflammation, and hypertrophy. IPA further predicted that the identified differentially expressed genes were the targets of 50 upstream regulators, including transcription factors (e.g., MYC and NRF1) and microRNAs (e.g., miR-335-3p and miR-338-3p). These data demonstrate that the vagus nerve has a major impact on the myocardial expression of genes involved in the regulation of key biological pathways. The transcriptional response of the ventricular myocardium induced by stimulation of vagal efferents is consistent with the beneficial effect of maintained/increased vagal activity on the heart.NEW & NOTEWORTHY This experimental animal study investigated the immediate transcriptional response of the ventricular myocardium to selective stimulation of vagal efferent activity. Vagal stimulation induced significant transcriptional changes in the heart involving the pathways controlling autonomic signaling, inflammation, fibrosis, and hypertrophy. This study provides the first direct evidence that myocardial gene expression is modulated by the activity of the autonomic nervous system.

Preoperative N-terminal pro-B-type natriuretic peptide and myocardial injury after stopping or continuing renin-angiotensin system inhibitors in noncardiac surgery: a prespecified analysis of a phase 2 randomised controlled multicentre trial.

BACKGROUND: Patients with elevated preoperative plasma N-terminal pro-B-type natriuretic peptide (NT-proBNP >100 pg ml-1) experience more complications after noncardiac surgery. Individuals prescribed renin-angiotensin system (RAS) inhibitors for cardiometabolic disease are at particular risk of perioperative myocardial injury and complications. We hypothesised that stopping RAS inhibitors before surgery increases the risk of perioperative myocardial injury, depending on preoperative risk stratified by plasma NT-proBNP concentrations. METHODS: In a preplanned analysis of a phase 2a trial in six UK centres, patients ≥60 yr old undergoing elective noncardiac surgery were randomly assigned either to stop or continue RAS inhibitors before surgery. The pharmacokinetic profile of individual RAS inhibitors determined for how long they were stopped before surgery. The primary outcome, masked to investigators, clinicians, and patients, was myocardial injury (plasma high-sensitivity troponin-T ≥15 ng L-1 or a ≥5 ng L-1 increase, when preoperative high-sensitivity troponin-T ≥15 ng L-1) within 48 h after surgery. The co-exposures of interest were preoperative plasma NT-proBNP (< or >100 pg ml -1) and stopping or continuing RAS inhibitors. RESULTS: Of 241 participants, 101 (41.9%; mean age 71 [7] yr; 48% females) had preoperative NT-proBNP >100 pg ml -1 (median 339 [160-833] pg ml-1), of whom 9/101 (8.9%) had a formal diagnosis of cardiac failure. Myocardial injury occurred in 63/101 (62.4%) subjects with NT-proBNP >100 pg ml-1, compared with 45/140 (32.1%) subjects with NT-proBNP <100 pg ml -1 {odds ratio (OR) 3.50 (95% confidence interval [CI] 2.05-5.99); P<0.0001}. For subjects with preoperative NT-proBNP <100 pg ml-1, 30/75 (40%) who stopped RAS inhibitors had myocardial injury, compared with 15/65 (23.1%) who continued RAS inhibitors (OR for stopping 2.22 [95% CI 1.06-4.65]; P=0.03). For preoperative NT-proBNP >100 pg ml-1, myocardial injury rates were similar regardless of stopping (62.2%) or continuing (62.5%) RAS inhibitors (OR for stopping 0.98 [95% CI 0.44-2.22]). CONCLUSIONS: Stopping renin-angiotensin system inhibitors in lower-risk patients (preoperative NT-proBNP <100 pg ml -1) increased the likelihood of myocardial injury before noncardiac surgery.

Interleukin-1 receptor antagonist, mode of analgesia and risk of Caesarean delivery after onset of labour: a Mendelian randomisation analysis.

BACKGROUND: Lower circulating levels of the anti-inflammatory cytokine interleukin-1 receptor antagonist (IL-1ra) are associated with intrapartum inflammation and epidural analgesia-related maternal fever, both of which increase the rate of obstetric interventions. We hypothesised that genetic variants determining IL-1ra levels would be associated with Caesarean delivery rates after the onset of labour. METHODS: We performed Mendelian randomisation analyses in parous women ≥16 yr old who received either non-neuraxial or neuraxial analgesia for their first two labours (UK Biobank). We used an established genetic score (calculated as 0-4, determined by the presence/absence of rs6743376 and rs1542176 alleles), in which the complete absence of both alleles causes the lowest IL-1ra levels. The primary outcome was Caesarean delivery after the onset of labour (odds ratio [OR]: 95% confidence intervals). RESULTS: There were 7731 women (mean [standard deviation] age at first birth: 25 [5] yr) who had complete genetic scores and delivery data. For women who received non-neuraxial analgesia, Caesarean delivery rates were different across allele scores (χ2=12.4; P=0.015): 104/596 (17.4%) women with zero allele score underwent Caesarean delivery, compared with 654/5015 (13.0%) with allele score ≥1 (OR 1.41; 1.12-1.77). For women who had neuraxial analgesia, Caesarean delivery was not different across allele scores, ranging from 18.1% to 20.8% (χ2=0.29; P=0.99). Caesarean delivery was independent of type of analgesia for 818/7731 (10.6%) women with zero allele scores (OR 0.93; 0.63-1.39), but was higher in women receiving neuraxial analgesia with allele scores ≥1 (OR 1.55; 1.35-1.79; P<0.001). CONCLUSIONS: Mendelian randomisation analysis suggests that higher IL-1ra levels are associated with reduced Caesarean delivery rate. Neuraxial analgesia appears to disrupt this link. CLINICAL TRIAL REGISTRATION: UK Biobank study 62745.

Cardiopulmonary exercise capacity and preoperative markers of inflammation.

Explanatory mechanisms for the association between poor exercise capacity and infections following surgery are underexplored. We hypothesized that aerobic fitness-assessed by cardiopulmonary exercise testing (CPET)-would be associated with circulating inflammatory markers, as quantified by the neutrophil-lymphocyte ratio (NLR) and monocyte subsets. The association between cardiopulmonary reserve and inflammation was tested by multivariable regression analysis with covariates including anaerobic threshold (AT) and malignancy. In a first cohort of 240 colorectal patients, AT was identified as the sole factor associated with higher NLR (P = 0.03) and absolute and relative lymphopenia (P = 0.01). Preoperative leukocyte subsets and monocyte CD14(+) expression (downregulated by endotoxin and indicative of chronic inflammation) were also assessed in two further cohorts of age-matched elective gastrointestinal and orthopaedic surgical patients. Monocyte CD14(+) expression was lower in gastrointestinal patients (n = 43) compared to age-matched orthopaedic patients (n = 31). The circulating CD14(+)CD16(-) monocyte subset was reduced in patients with low cardiopulmonary reserve. Poor exercise capacity in patients without a diagnosis of heart failure is independently associated with markers of inflammation. These observations suggest that preoperative inflammation associated with impaired cardiorespiratory performance may contribute to the pathophysiology of postoperative outcome.

Human neutrophils communicate remotely via calcium-dependent glutamate-induced glutamate release.

Neutrophils are white blood cells that are critical to acute inflammatory and adaptive immune responses. Their swarming-pattern behavior is controlled by multiple cellular cascades involving calcium-dependent release of various signaling molecules. Previous studies have reported that neutrophils express glutamate receptors and can release glutamate but evidence of direct neutrophil-neutrophil communication has been elusive. Here, we hold semi-suspended cultured human neutrophils in patch-clamp whole-cell mode to find that calcium mobilization induced by stimulating one neutrophil can trigger an N-methyl-D-aspartate (NMDA) receptor-driven membrane current and calcium signal in neighboring neutrophils. We employ an enzymatic-based imaging assay to image, in real time, glutamate release from neutrophils induced by glutamate released from their neighbors. These observations provide direct evidence for a positive-feedback inter-neutrophil communication that could contribute to mechanisms regulating communal neutrophil behavior.

Transauricular nerve stimulation in acute ischaemic stroke requiring mechanical thrombectomy: Protocol for a phase 2A, proof-of-concept, sham-controlled randomised trial.

BACKGROUND: Labile blood pressure after acute ischaemic stroke requiring mechanical thrombectomy is independently associated with poor patient outcomes. OBJECTIVES: This study protocol describes is designed to determine whether transauricular nerve stimulation, improves baroreflex sensitivity, reduces blood pressure variability in the first 24 hours after acute ischaemic stroke requiring mechanical thrombectomy. DESIGN: PHASE 2A, PROOF-OF-CONCEPT, SHAM-CONTROLLED RANDOMISED TRIAL: Methods and Analysis: 36 individuals undergoing mechanical thrombectomy for acute ischaemic stroke with established hypertension aged >18 years will be randomly allocated to receive bilateral active or sham transauricular nerve stimulation for the duration of the mechanical thrombectomy procedure (AffeX-CT/001 investigational device). The intervention will be repeated for 1h the morning following the mechanical thrombectomy. Non-invasive blood pressure will be measured ≥2h for 24h after mechanical thrombectomy. Holter electrocardiographic monitoring will be recorded during transauricular nerve stimulation. Participants, clinicians and investigators will be masked to treatment allocations. The primary outcome will be the coefficient of variation of systolic blood pressure. Secondary outcomes include additional estimates of blood pressure variability and time/frequency-domain measures of autonomic cardiac modulation An adjusted sample size of 36 patients is required to have a 90% chance of detecting, as significant at the 5% level, a difference in the coefficient of variation in systolic blood pressure of 5±4mmHg between sham and active stimulation [assuming 5% non-compliance rate in each group]. Ethics: confirmed on 16 March 2023 by HRA and Health and Care Research Wales ethics committee (reference 23/WA/0013). DISCUSSION: This study will provide proof-of-concept data that examines whether non-invasive autonomic neuromodulation can be used to favourably modify blood pressure and autonomic control after acute ischaemic stroke requiring mechanical thrombectomy. TRIAL REGISTRATION: Trial registration number: NCT05417009.

Potent phagocytic activity with impaired antigen presentation identifying lipopolysaccharide-tolerant human monocytes: demonstration in isolated monocytes from cystic fibrosis patients.

Monocyte exposure to LPS induces a transient state in which these cells are refractory to further endotoxin stimulation. This phenomenon, termed endotoxin tolerance (ET), is characterized by a decreased production of cytokines in response to the proinflammatory stimulus. We have established a robust model of ET and have determined the time frame and features of LPS unresponsiveness in cultured human monocytes. A large number of genes transcribed in tolerant monocytes were classified as either "tolerizable" or "nontolerizable" depending on their expression levels during the ET phase. Tolerant monocytes exhibit rapid IL-1R-associated kinase-M (IRAK-M) overexpression, high levels of triggering receptor expressed on myeloid cells-1 (TREM-1) and CD64, and a marked down-regulation of MHC molecules and NF-kappaB2. These cells combine potent phagocytic activity with impaired capability for Ag presentation. We also show that circulating monocytes isolated from cystic fibrosis patients share all the determinants that characterize cells locked in an ET state. These findings identify a new mechanism that contributes to impaired inflammation in cystic fibrosis patients despite a high frequency of infections. Our results indicate that a tolerant phenotype interferes with timing, efficiency, and outcome of the innate immune responses against bacterial infections.

Early growth response 2 (EGR2) is a novel regulator of the senescence programme.

Senescence, a state of stable growth arrest, plays an important role in ageing and age-related diseases in vivo. Although the INK4/ARF locus is known to be essential for senescence programmes, the key regulators driving p16 and ARF transcription remain largely underexplored. Using siRNA screening for modulators of the p16/pRB and ARF/p53/p21 pathways in deeply senescent human mammary epithelial cells (DS HMECs) and fibroblasts (DS HMFs), we identified EGR2 as a novel regulator of senescence. EGR2 expression is up-regulated during senescence, and its ablation by siRNA in DS HMECs and HMFs transiently reverses the senescent phenotype. We demonstrate that EGR2 activates the ARF and p16 promoters and directly binds to both the ARF and p16 promoters. Loss of EGR2 down-regulates p16 levels and increases the pool of p16- p21- 'reversed' cells in the population. Moreover, EGR2 overexpression is sufficient to induce senescence. Our data suggest that EGR2 is a direct transcriptional activator of the p16/pRB and ARF/p53/p21 pathways in senescence and a novel marker of senescence.

Low-molecular-weight polyethylene glycol improves survival in experimental sepsis.

OBJECTIVE: For several chronic inflammatory disease states, therapy is enhanced by improving the pharmacokinetic properties of anti-inflammatory drugs through conjugation with polyethylene glycol. We hypothesized that part of the beneficial action of PEGylated drugs may be derived from the anti-inflammatory properties of polyethylene glycol (PEG) itself. DESIGN: Randomized, double-blinded, controlled ex vivo and in vivo laboratory studies. SETTING: University research laboratories. SUBJECTS: Human neutrophils and mononuclear cells, macrophage cell line, and adult rats and mice. INTERVENTIONS: The effect of PEG (either low-molecular-weight [200-400] or high-molecular-weight [>4000]) was assessed on survival after systemic inflammation induced by lipopolysaccharide or zymosan. The effects of PEG on zymosan, lipopolysaccharide, or streptolysin-induced inflammatory and bioenergetic responses of immune cells were also assessed. MEASUREMENTS AND MAIN RESULTS: Low-molecular-weight PEG reduced inflammatory cytokine expression, pyrexia, and mortality by >50% in both lipopolysaccharide and zymosan models of sepsis. Low-molecular-weight PEG reduced cytokine expression both in vivo and in vitro, and attenuated activation of human neutrophils in response to lipopolysaccharide or zymosan. By contrast, high-molecular-weight PEG conferred less significant survival effects after lipopolysaccharide and zymosan, and it did not exhibit such profound anti-inflammatory effects. Low-molecular-weight PEG attenuated lipopolysaccharide-induced activation of pro-apoptotic pathways (lysophosphatidic acid receptor and caspase-domain signaling) in the livers of endotoxemic rats. Streptolysin-induced necrosis of human neutrophils was reduced by low-molecular-weight PEG, indicating a mechanism that involves coating and/or stabilizing the cellular membrane. Low-molecular-weight PEG preserved human neutrophil responses to septic serum and bioenergetic function in macrophages and neutrophils. CONCLUSION: PEG is a commonly used, safe, nonimmunogenic molecule possessing hitherto unappreciated anti-inflammatory properties. Low-molecular-weight PEG may potentially play a role in the therapy of systemic inflammation and sepsis.

PINK1 deficiency in ß-cells increases basal insulin secretion and improves glucose tolerance in mice.

The Parkinson's disease (PD) gene, PARK6, encodes the PTEN-induced putative kinase 1 (PINK1) mitochondrial kinase, which provides protection against oxidative stress-induced apoptosis. Given the link between glucose metabolism, mitochondrial function and insulin secretion in ß-cells, and the reported association of PD with type 2 diabetes, we investigated the response of PINK1-deficient ß-cells to glucose stimuli to determine whether loss of PINK1 affected their function. We find that loss of PINK1 significantly impairs the ability of mouse pancreatic ß-cells (MIN6 cells) and primary intact islets to take up glucose. This was accompanied by higher basal levels of intracellular calcium leading to increased basal levels of insulin secretion under low glucose conditions. Finally, we investigated the effect of PINK1 deficiency in vivo and find that PINK1 knockout mice have improved glucose tolerance. For the first time, these combined results demonstrate that loss of PINK1 function appears to disrupt glucose-sensing leading to enhanced insulin release, which is uncoupled from glucose uptake, and suggest a key role for PINK1 in ß-cell function.

A functional screen for regulators of CKDN2A reveals MEOX2 as a transcriptional activator of INK4a.

The CDKN2A locus encodes two important tumor suppressors, INK4a and ARF, which respond to oncogenic stresses by inducing cellular senescence. We conducted a genome-scale cDNA overexpression screen using a reporter containing INK4a regulatory sequences to identify novel transcriptional activators of this locus. This screen revealed 285 cDNAs that putatively regulate the transcriptional activation of INK4a. Of these, 56 are annotated as transcription factors, including two previously reported activators of the locus, ETS2 and JUNB. Fourteen genes were further validated for activity and specificity, including several homeodomain proteins. We found that the transcription of one of these, the homeodomain protein MEOX2 (GAX) is enhanced in primary cells during the induction of senescence, and forced expression of this protein results in the induction of premature senescence. We further demonstrate that MEOX2-induced senescence is dependent upon INK4a activity, and chromatin immunoprecipitation studies indicate that MEOX2 directly binds the INK4a promoter. These results support a role for this homeodomain protein as a direct regulator of INK4a transcription and senescence in human cells.

Contribution of p16(INK4a) to replicative senescence of human fibroblasts.

In standard conditions of tissue culture, human fibroblasts undergo a limited number of population doublings before entering a state of irreversible growth arrest termed replicative senescence or M1. The arrest is triggered by a combination of telomere dysfunction and the stresses inflicted by culture conditions and is implemented, at least in part, by the cyclin-dependent kinase inhibitors p21(CIP1) and p16(INK4a). To investigate the role of p16(INK4a), we have studied fibroblasts from members of melanoma prone kindreds with mutations in one or both copies of the CDKN2A locus. The mutations affect the function of p16(INK4a) but not of the alternative product, p14(ARF). The p16(INK4a)-defective fibroblasts have an above average life span, compared to the heterozygous and normal age-matched controls, but they arrest with characteristics typical of senescence. Using agents that are known to bypass M1, such as DNA tumor virus oncoproteins or the Bmi1 transcriptional repressor, we provide evidence that p16(INK4a) defective cells arrest at a stage that is operationally between M1 and M2 (crisis). As well as indicating that p16(INK4a) contributes to but is not essential for replicative senescence of human fibroblasts, our data reveal considerable heterogeneity in the levels and accumulation of p16(INK4a) in different strains.