Transcriptional profile of the rat cardiovascular system at single cell resolution.

Background: Despite the critical role of the cardiovascular system, our understanding of its cellular and transcriptional diversity remains limited. We therefore sought to characterize the cellular composition, phenotypes, molecular pathways, and communication networks between cell types at the tissue and sub-tissue level across the cardiovascular system of the healthy Wistar rat, an important model in preclinical cardiovascular research. We obtained high quality tissue samples under controlled conditions that reveal a level of cellular detail so far inaccessible in human studies. Methods and Results: We performed single nucleus RNA-sequencing in 78 samples in 10 distinct regions including the four chambers of the heart, ventricular septum, sinoatrial node, atrioventricular node, aorta, pulmonary artery, and pulmonary veins (PV), which produced an aggregate map of 505,835 nuclei. We identified 26 distinct cell types and additional subtypes, including a number of rare cell types such as PV cardiomyocytes and non-myelinating Schwann cells (NMSCs), and unique groups of vascular smooth muscle cells (VSMCs), endothelial cells (ECs) and fibroblasts (FBs), which gave rise to a detailed cell type distribution across tissues. We demonstrated differences in the cellular composition across different cardiac regions and tissue-specific differences in transcription for each cell type, highlighting the molecular diversity and complex tissue architecture of the cardiovascular system. Specifically, we observed great transcriptional heterogeneities among ECs and FBs. Importantly, several cell subtypes had a unique regional localization such as a subtype of VSMCs enriched in the large vasculature. We found the cellular makeup of PV tissue is closer to heart tissue than to the large arteries. We further explored the ligand-receptor repertoire across cell clusters and tissues, and observed tissue-enriched cellular communication networks, including heightened Nppa - Npr1/2/3 signaling in the sinoatrial node. Conclusions: Through a large single nucleus sequencing effort encompassing over 500,000 nuclei, we broadened our understanding of cellular transcription in the healthy cardiovascular system. The existence of tissue-restricted cellular phenotypes suggests regional regulation of cardiovascular physiology. The overall conservation in gene expression and molecular pathways across rat and human cell types, together with our detailed transcriptional characterization of each cell type, offers the potential to identify novel therapeutic targets and improve preclinical models of cardiovascular disease.

Unsupervised removal of systematic background noise from droplet-based single-cell experiments using CellBender.

Droplet-based single-cell assays, including single-cell RNA sequencing (scRNA-seq), single-nucleus RNA sequencing (snRNA-seq) and cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq), generate considerable background noise counts, the hallmark of which is nonzero counts in cell-free droplets and off-target gene expression in unexpected cell types. Such systematic background noise can lead to batch effects and spurious differential gene expression results. Here we develop a deep generative model based on the phenomenology of noise generation in droplet-based assays. The proposed model accurately distinguishes cell-containing droplets from cell-free droplets, learns the background noise profile and provides noise-free quantification in an end-to-end fashion. We implement this approach in the scalable and robust open-source software package CellBender. Analysis of simulated data demonstrates that CellBender operates near the theoretically optimal denoising limit. Extensive evaluations using real datasets and experimental benchmarks highlight enhanced concordance between droplet-based single-cell data and established gene expression patterns, while the learned background noise profile provides evidence of degraded or uncaptured cell types.

Single-nucleus profiling of human dilated and hypertrophic cardiomyopathy.

Heart failure encompasses a heterogeneous set of clinical features that converge on impaired cardiac contractile function1,2 and presents a growing public health concern. Previous work has highlighted changes in both transcription and protein expression in failing hearts3,4, but may overlook molecular changes in less prevalent cell types. Here we identify extensive molecular alterations in failing hearts at single-cell resolution by performing single-nucleus RNA sequencing of nearly 600,000 nuclei in left ventricle samples from 11 hearts with dilated cardiomyopathy and 15 hearts with hypertrophic cardiomyopathy as well as 16 non-failing hearts. The transcriptional profiles of dilated or hypertrophic cardiomyopathy hearts broadly converged at the tissue and cell-type level. Further, a subset of hearts from patients with cardiomyopathy harbour a unique population of activated fibroblasts that is almost entirely absent from non-failing samples. We performed a CRISPR-knockout screen in primary human cardiac fibroblasts to evaluate this fibrotic cell state transition; knockout of genes associated with fibroblast transition resulted in a reduction of myofibroblast cell-state transition upon TGFß1 stimulation for a subset of genes. Our results provide insights into the transcriptional diversity of the human heart in health and disease as well as new potential therapeutic targets and biomarkers for heart failure.

TAILS Identifies Candidate Substrates and Biomarkers of ADAMTS7, a Therapeutic Protease Target in Coronary Artery Disease.

Loss-of-function mutations in the secreted enzyme ADAMTS7 (a disintegrin and metalloproteinase with thrombospondin motifs 7) are associated with protection for coronary artery disease. ADAMTS7 catalytic inhibition has been proposed as a therapeutic strategy for treating coronary artery disease; however, the lack of an endogenous substrate has hindered the development of activity-based biomarkers. To identify ADAMTS7 extracellular substrates and their cleavage sites relevant to vascular disease, we used TAILS (terminal amine isotopic labeling of substrates), a method for identifying protease-generated neo-N termini. We compared the secreted proteome of vascular smooth muscle and endothelial cells expressing either full-length mouse ADAMTS7 WT, catalytic mutant ADAMTS7 E373Q, or a control luciferase adenovirus. Significantly enriched N-terminal cleavage sites in ADAMTS7 WT samples were compared to the negative control conditions and filtered for stringency, resulting in catalogs of high confidence candidate ADAMTS7 cleavage sites from our three independent TAILS experiments. Within the overlap of these discovery sets, we identified 24 unique cleavage sites from 16 protein substrates, including cleavage sites in EFEMP1 (EGF-containing fibulin-like extracellular matrix protein 1/Fibulin-3). The ADAMTS7 TAILS preference for EFEMP1 cleavage at the amino acids 123.124 over the adjacent 124.125 site was validated using both endogenous EFEMP1 and purified EFEMP1 in a binary in vitro cleavage assay. Collectively, our TAILS discovery experiments have uncovered hundreds of potential substrates and cleavage sites to explore disease-related biological substrates and facilitate activity-based ADAMTS7 biomarker development.

Vascular smooth muscle cell phenotype switching in carotid atherosclerosis.

Carotid plaque instability contributes to large vessel ischemic stroke. Although vascular smooth muscle cells (VSMCs) affect atherosclerotic growth and instability, no treatments aimed at improving VSMC function are available. Large genetic studies investigating atherosclerosis and carotid disease in relation to the risk of stroke have implicated polymorphisms at the HDAC9 locus. The HDAC9 protein has been shown to affect the VSMC phenotype; however, how this might affect carotid disease is unknown. We conducted a pilot investigation using single nuclei RNA sequencing of human carotid tissue to identify cells expressing HDAC9 and specifically investigate the role of the HDAC9 in carotid atherosclerosis. We found that carotid VSMCs express HDAC9 and genes typically associated with immune characteristics. Using cellular assays, we have demonstrated that recruitment of macrophages can be modulated by HDAC9 expression. HDAC9 expression might affect carotid plaque stability and progression through its effects on the VSMC phenotype and recruitment of immune cells.

Endothelial ARHGEF26 is an angiogenic factor promoting VEGF signalling.

AIMS: Genetic studies have implicated the ARHGEF26 locus in the risk of coronary artery disease (CAD). However, the causal pathways by which DNA variants at the ARHGEF26 locus confer risk for CAD are incompletely understood. We sought to elucidate the mechanism responsible for the enhanced risk of CAD associated with the ARHGEF26 locus. METHODS AND RESULTS: In a conditional analysis of the ARHGEF26 locus, we show that the sentinel CAD-risk signal is significantly associated with various non-lipid vascular phenotypes. In human endothelial cell (EC), ARHGEF26 promotes the angiogenic capacity, and interacts with known angiogenic factors and pathways. Quantitative mass spectrometry showed that one CAD-risk coding variant, rs12493885 (p.Val29Leu), resulted in a gain-of-function ARHGEF26 that enhances proangiogenic signalling and displays enhanced interactions with several proteins partially related to the angiogenic pathway. ARHGEF26 is required for endothelial angiogenesis by promoting macropinocytosis of Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) on cell membrane and is crucial to Vascular Endothelial Growth Factor (VEGF)-dependent murine vessel sprouting ex vivo. In vivo, global or tissue-specific deletion of ARHGEF26 in EC, but not in vascular smooth muscle cells, significantly reduced atherosclerosis in mice, with enhanced plaque stability. CONCLUSIONS: Our results demonstrate that ARHGEF26 is involved in angiogenesis signaling, and that DNA variants within ARHGEF26 that are associated with CAD risk could affect angiogenic processes by potentiating VEGF-dependent angiogenesis.

Deep learning enables genetic analysis of the human thoracic aorta.

Enlargement or aneurysm of the aorta predisposes to dissection, an important cause of sudden death. We trained a deep learning model to evaluate the dimensions of the ascending and descending thoracic aorta in 4.6 million cardiac magnetic resonance images from the UK Biobank. We then conducted genome-wide association studies in 39,688 individuals, identifying 82 loci associated with ascending and 47 with descending thoracic aortic diameter, of which 14 loci overlapped. Transcriptome-wide analyses, rare-variant burden tests and human aortic single nucleus RNA sequencing prioritized genes including SVIL, which was strongly associated with descending aortic diameter. A polygenic score for ascending aortic diameter was associated with thoracic aortic aneurysm in 385,621 UK Biobank participants (hazard ratio = 1.43 per s.d., confidence interval 1.32-1.54, P = 3.3 × 10-20). Our results illustrate the potential for rapidly defining quantitative traits with deep learning, an approach that can be broadly applied to biomedical images.

Coronary Disease Association With ADAMTS7 Is Due to Protease Activity.

[Figure: see text].

Myocyte Specific Upregulation of ACE2 in Cardiovascular Disease: Implications for SARS-CoV-2 mediated myocarditis.

Coronavirus disease 2019 (COVID-19) is a global pandemic caused by a novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). SARS-CoV-2 infection of host cells occurs predominantly via binding of the viral surface spike protein to the human angiotensin-converting enzyme 2 (ACE2) receptor. Hypertension and pre-existing cardiovascular disease are risk factors for morbidity from COVID-19, and it remains uncertain whether the use of angiotensin converting enzyme inhibitors (ACEi) or angiotensin receptor blockers (ARB) impacts infection and disease. Here, we aim to shed light on this question by assessing ACE2 expression in normal and diseased human myocardial samples profiled by bulk and single nucleus RNA-seq.

Transcriptional and Cellular Diversity of the Human Heart.

BACKGROUND: The human heart requires a complex ensemble of specialized cell types to perform its essential function. A greater knowledge of the intricate cellular milieu of the heart is critical to increase our understanding of cardiac homeostasis and pathology. As recent advances in low-input RNA sequencing have allowed definitions of cellular transcriptomes at single-cell resolution at scale, we have applied these approaches to assess the cellular and transcriptional diversity of the nonfailing human heart. METHODS: Microfluidic encapsulation and barcoding was used to perform single nuclear RNA sequencing with samples from 7 human donors, selected for their absence of overt cardiac disease. Individual nuclear transcriptomes were then clustered based on transcriptional profiles of highly variable genes. These clusters were used as the basis for between-chamber and between-sex differential gene expression analyses and intersection with genetic and pharmacologic data. RESULTS: We sequenced the transcriptomes of 287 269 single cardiac nuclei, revealing 9 major cell types and 20 subclusters of cell types within the human heart. Cellular subclasses include 2 distinct groups of resident macrophages, 4 endothelial subtypes, and 2 fibroblast subsets. Comparisons of cellular transcriptomes by cardiac chamber or sex reveal diversity not only in cardiomyocyte transcriptional programs but also in subtypes involved in extracellular matrix remodeling and vascularization. Using genetic association data, we identified strong enrichment for the role of cell subtypes in cardiac traits and diseases. Intersection of our data set with genes on cardiac clinical testing panels and the druggable genome reveals striking patterns of cellular specificity. CONCLUSIONS: Using large-scale single nuclei RNA sequencing, we defined the transcriptional and cellular diversity in the normal human heart. Our identification of discrete cell subtypes and differentially expressed genes within the heart will ultimately facilitate the development of new therapeutics for cardiovascular diseases.

Dietary protein restriction reduces circulating VLDL triglyceride levels via CREBH-APOA5-dependent and -independent mechanisms.

Hypertriglyceridemia is an independent risk factor for cardiovascular disease. Dietary interventions based on protein restriction (PR) reduce circulating triglycerides (TGs), but underlying mechanisms and clinical relevance remain unclear. Here, we show that 1 week of a protein-free diet without enforced calorie restriction significantly lowered circulating TGs in both lean and diet-induced obese mice. Mechanistically, the TG-lowering effect of PR was due, in part, to changes in very low-density lipoprotein (VLDL) metabolism both in liver and peripheral tissues. In the periphery, PR stimulated VLDL-TG consumption by increasing VLDL-bound APOA5 expression and promoting VLDL-TG hydrolysis and clearance from circulation. The PR-mediated increase in Apoa5 expression was controlled by the transcription factor CREBH, which coordinately regulated hepatic expression of fatty acid oxidation-related genes, including Fgf21 and Ppara. The CREBH-APOA5 axis activation upon PR was intact in mice lacking the GCN2-dependent amino acid-sensing arm of the integrated stress response. However, constitutive hepatic activation of the amino acid-responsive kinase mTORC1 compromised CREBH activation, leading to blunted APOA5 expression and PR-recalcitrant hypertriglyceridemia. PR also contributed to hypotriglyceridemia by reducing the rate of VLDL-TG secretion, independently of activation of the CREBH-APOA5 axis. Finally, a randomized controlled clinical trial revealed that 4-6 weeks of reduced protein intake (7%-9% of calories) decreased VLDL particle number, increased VLDL-bound APOA5 expression, and lowered plasma TGs, consistent with mechanistic conservation of PR-mediated hypotriglyceridemia in humans with translational potential as a nutraceutical intervention for dyslipidemia.

Amino Acid Restriction Triggers Angiogenesis via GCN2/ATF4 Regulation of VEGF and H2S Production.

Angiogenesis, the formation of new blood vessels by endothelial cells (ECs), is an adaptive response to oxygen/nutrient deprivation orchestrated by vascular endothelial growth factor (VEGF) upon ischemia or exercise. Hypoxia is the best-understood trigger of VEGF expression via the transcription factor HIF1α. Nutrient deprivation is inseparable from hypoxia during ischemia, yet its role in angiogenesis is poorly characterized. Here, we identified sulfur amino acid restriction as a proangiogenic trigger, promoting increased VEGF expression, migration and sprouting in ECs in vitro, and increased capillary density in mouse skeletal muscle in vivo via the GCN2/ATF4 amino acid starvation response pathway independent of hypoxia or HIF1α. We also identified a requirement for cystathionine-γ-lyase in VEGF-dependent angiogenesis via increased hydrogen sulfide (H2S) production. H2S mediated its proangiogenic effects in part by inhibiting mitochondrial electron transport and oxidative phosphorylation, resulting in increased glucose uptake and glycolytic ATP production.

An Eighteen-Minute Submaximal Exercise Test to Assess Cardiac Fitness in Response to Aerobic Training.

Romagnoli, M, Alis, R, Sanchis-Gomar, F, Lippi, G, and Arduini, A. An 18-minute submaximal exercise test to assess cardiac fitness in response to aerobic training. J Strength Cond Res 32(10): 2846-2852, 2018-We aimed to evaluate the utility of a submaximal heart rate recovery (HRR) test to monitor changes in cardiac fitness after aerobic training. Twenty healthy subjects were assigned to a control (n = 10) or a training (n = 10) group. Subjects in the training group performed 8 weeks of bicycle training, followed by 8 weeks of detraining. Heart rate recovery was assessed after exercises at 65% and 80% HRmax. The HRR test was performed at weeks 0 (W0), 4 (W4), 8 (W8), and 16 (W16) in the training group and at W0 and W8 in the control group. Heart rate recovery indices changed in response to training and detraining. Absolute HRR at 60, 120, and 180 seconds after exercise increased at both exercise intensities at W8 of training (p < 0.01, W8 vs. W0) and returned to the pretraining level after detraining (p > 0.05, W16 vs. W0). Time constants of fast HRR recovery (<1 minute) changed with training (p < 0.05-0.01, W8 vs. W0) and detraining (p > 0.05, W16 vs. W0) but only at 65% HRmax. At the end of the 3-minute recovery period, the predicted heart rate (HR) value (A0) and the HR recovered (Amax) from the monoexponential analysis changed with training (p < 0.05-0.01, W8 vs. W0) and detraining (p > 0.05, W16 vs. W0). We conclude that this novel submaximal HRR test is highly sensitive for monitoring cardiac fitness during training and detraining in healthy people. Because this test is simple, inexpensive, and the data are reliable and easy to analyze, we hope that it may be of interest to the sports science community.

NRF1 Is an ER Membrane Sensor that Is Central to Cholesterol Homeostasis.

Cholesterol is a critical nutrient requiring tight constraint in the endoplasmic reticulum (ER) due to its uniquely challenging biophysical properties. While the mechanisms by which the ER defends against cholesterol insufficiency are well described, it remains unclear how the ER senses and effectively defends against cholesterol excess. Here, we identify the ER-bound transcription factor nuclear factor erythroid 2 related factor-1, Nrf1/Nfe2L1, as a critical mediator of this process. We show that Nrf1 directly binds to and specifically senses cholesterol in the ER through a defined domain and that cholesterol regulates Nrf1 turnover, processing, localization, and activity. In Nrf1 deficiency, in vivo cholesterol challenges induce massive hepatic cholesterol accumulation and damage, which is rescued by replacing Nrf1 exogenously. This Nrf1-mediated mechanism involves the suppression of CD36-driven inflammatory signaling and derepression of liver X receptor activity. These findings reveal Nrf1 as a guardian of cholesterol homeostasis and a core component of adaptive responses to excess cellular cholesterol.

Relaxant Effects of the Selective Estrogen Receptor Modulator, Bazedoxifene, and Estrogen Receptor Agonists in Isolated Rabbit Basilar Artery.

We have previously shown that the selective estrogen receptor modulator, bazedoxifene, improves the consequences of ischemic stroke. Now we aimed to characterize the effects and mechanisms of action of bazedoxifene in cerebral arteries. Male rabbit isolated basilar arteries were used for isometric tension recording and quantitative polymerase chain reaction. Bazedoxifene relaxed cerebral arteries, as 17-ß-estradiol, 4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol [estrogen receptor (ER) α agonist], and G1 [G protein-coupled ER (GPER) agonist] did it (4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol > bazedoxifene = G1 > 17-ß-estradiol). 2,3-Bis(4-hydroxyphenyl)-propionitrile (ERß agonist) had no effect. Expression profile of genes encoding for ERα (ESR1), ERß (ESR2), and GPER was GPER > ESR1 > ESR2. As to the endothelial mechanisms, endothelium removal, N-nitro-L-arginine methyl ester, and indomethacin, did not modify the relaxant responses to bazedoxifene. As to the K channels, both a high-K medium and the Kv blocker, 4-aminopyridine, inhibited the bazedoxifene-induced relaxations, whereas tetraethylammonium (nonselective K channel blocker), glibenclamide (selective KATP blocker) or iberiotoxin (selective KCa blocker) were without effect. Bazedoxifene also inhibited both Ca- and Bay K8644-elicited contractions. Therefore, bazedoxifene induces endothelium-independent relaxations of cerebral arteries through (1) activation of GPER and ERα receptors; (2) increase of K conductance through Kv channels; and (3) inhibition of Ca entry through L-type Ca channels. Such a profile is compatible with the beneficial effects of estrogenic compounds (eg, SERMs) on vascular function and, specifically, that concerning the brain. Therefore, bazedoxifene could be useful in the treatment of cerebral disorders in which the cerebrovascular function is compromised (eg, stroke).

METABOLISM. S-Nitrosylation links obesity-associated inflammation to endoplasmic reticulum dysfunction.

The association between inflammation and endoplasmic reticulum (ER) stress has been observed in many diseases. However, if and how chronic inflammation regulates the unfolded protein response (UPR) and alters ER homeostasis in general, or in the context of chronic disease, remains unknown. Here, we show that, in the setting of obesity, inflammatory input through increased inducible nitric oxide synthase (iNOS) activity causes S-nitrosylation of a key UPR regulator, IRE1α, which leads to a progressive decline in hepatic IRE1α-mediated XBP1 splicing activity in both genetic (ob/ob) and dietary (high-fat diet-induced) models of obesity. Finally, in obese mice with liver-specific IRE1α deficiency, reconstitution of IRE1α expression with a nitrosylation-resistant variant restored IRE1α-mediated XBP1 splicing and improved glucose homeostasis in vivo. Taken together, these data describe a mechanism by which inflammatory pathways compromise UPR function through iNOS-mediated S-nitrosylation of IRE1α, which contributes to defective IRE1α activity, impaired ER function, and prolonged ER stress in obesity.

Metabolic adaptation and neuroprotection differ in the retina and choroid in a piglet model of acute postnatal hypoxia.

BACKGROUND: Hypoxic-ischemic insults to the neonatal brain may cause neurodevelopmental disorders. Vulnerability of different areas of the neural tissue to hypoxic-ischemic stress might be explained by either heterogeneous sensitivity to oxygen or neuroprotective capability. Our understanding of regional heterogeneity is still incomplete in terms of metabolic reconfiguration and/or activation of neuroprotective mechanisms. METHODS: We studied, by western blotting, reverse-transcriptase PCR, and tandem mass spectrometry, the response of retina and choroid at protein, gene, and metabolic levels during hypoxia in a piglet model of acute postnatal hypoxia. RESULTS: We evidenced a metabolic shift towards glycolysis in choroid after hypoxia while retina experienced a dramatic energy stress with decreased mitochondrial metabolites. Hypoxia-inducible transcription factor-1α (HIF-1α) was not stabilized in retina during hypoxia, supported by a deficient signaling from v-akt murine thymoma viral oncogene (AKT) and ERK1/2, and unchanged glutathione redox status. In retina, but not in choroid, phosphorylation of p65 (NF-κB) and increased transcription of target genes may have a major role during hypoxic stress. CONCLUSION: We showed that the retina engages a distinct pattern of signaling and transcriptional events than observed in the choroid. Retina and choroid may reflect regional sensitivity to hypoxia. While prolonged and intense hypoxia may jeopardize retinal cell survival, choroid sets up a different pattern of response, which promotes adaptation to these adverse conditions.

Small-molecule inhibitors of PKR improve glucose homeostasis in obese diabetic mice.

Obesity and metabolic diseases appear as clusters, often featuring high risk for insulin resistance and type 2 diabetes, and constitute a major global health problem with limited treatment options. Previous studies have shown that double-stranded RNA-dependent kinase, PKR, plays an important role in the nutrient/pathogen-sensing interface, and acts as a key modulator of chronic metabolic inflammation, insulin sensitivity, and glucose homeostasis in obesity. Recently, pathological PKR activation was also demonstrated in obese humans, strengthening its prospects as a potential drug target. Here, we investigate the use of two structurally distinct small-molecule inhibitors of PKR in the treatment of insulin resistance and type 2 diabetes in cells and in a mouse model of severe obesity and insulin resistance. Inhibition of PKR reduced stress-induced Jun NH2-terminal kinase activation and insulin receptor substrate 1 serine phosphorylation in vitro and in vivo. In addition, treatment with both PKR inhibitors reduced adipose tissue inflammation, improved insulin sensitivity, and improved glucose intolerance in mice after the establishment of obesity and insulin resistance. Our findings suggest that pharmacologically targeting PKR may be an effective therapeutic strategy for the treatment of insulin resistance and type 2 diabetes.

Metabolomic Analysis of the Effect of Postnatal Hypoxia on the Retina in a Newly Born Piglet Model.

The availability of reliable biomarkers of brain injury secondary to birth asphyxia could substantially improve clinical grading, therapeutic intervention strategies, and prognosis. In this study, changes in the metabolome of retinal tissue caused by profound hypoxia in an established neonatal piglet model were investigated using an ultra performance liquid chromatography - quadrupole time of flight mass spectrometry (UPLC-QTOFMS) untargeted metabolomic approach, which included Partial Least Squares - Discriminant Analysis (PLSDA) multivariate data analysis. The initial identification of a set of discriminant metabolites from UPLC-QTOFMS data was confirmed by target UPLC-MS/MS and allowed the selection of endogenous CDP-choline as a promising candidate biomarker for hypoxia-derived brain damage assessing intensity of retinal hypoxia. Results from this study will foster further research on CDP-choline changes occurring during resuscitation.