Biology subjects

Chu, A.

Publications and source records attributed to Chu, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Comprehensive characterization of genomic, transcriptomic and epigenomic artifacts introduced in formalin-fixed, paraffin-embedded tissues.

Genomic, transcriptomic and epigenomic characterization has accelerated the discovery of clinically-relevant alterations in cancer, predominantly using fresh frozen (FF) specimens. However, clinical molecular pathology laboratories prefer formalin-fixed paraffin-embedded (FFPE) methods, known to introduce artifacts at the nucleic acid level, over fresh frozen methods. Extending the multi-platform analysis to FFPE specimens for comprehensive clinical molecular diagnosis requires a thorough understanding of the consequence of formalin-fixation. We present a detailed multi-platform characterization of FFPE preservation using paired FF specimens as the 'gold standard'. DNA and RNA were obtained from 38 patients across 6 cancer types using a FFPE optimized co-isolation. The impact of FFPE on exome sequencing was dependent on filtering, where a minimum coverage or supporting read filter can mitigate FFPE-specific false positives. Copy number alterations, MSI assessment, mutational signatures, and DNA methylation were comparable between FFPE and FF. FFPE biases in RNA expression can be overcome when using biology-relevant genes and we describe a novel consequence of FFPE on miRNA species diversity. Collectively, this data provides a broad view of FFPE artifact and offers best practices for overcome these biases.

bioinformatics↗

bioRxiv2026

Prenatal Stress Differentially Shapes Adult Behavior in Male and Female Offspring

BackgroundPrenatal stress (PNS) is a well-established risk factor for neuropsychiatric vulnerability, yet its sex-specific behavioral consequences remain incompletely defined. Because males and females follow distinct neurodevelopmental trajectories, clarifying how early-life stress differentially shapes behavior is essential for developing targeted interventions. However, few preclinical studies directly compare male and female offspring within the same experimental framework, limiting the ability to identify true sex-dependent effects. MethodsUsing a validated mouse model of gestational restraint stress, we conducted a comprehensive, within-study assessment of sex-dependent behavioral outcomes in adult offspring. Behavioral domains included locomotor activity, anxiety-like behavior, sociability, fear learning and extinction, recognition memory, and alcohol-related responses (ethanol preference and behavioral sensitivity), all measured using identical paradigms across sexes. ResultsPNS broadly disrupted behavior and cognition in both sexes, increasing locomotor activity and anxiety-like behavior, impairing fear extinction and recognition memory, and altering behavioral sensitivity to ethanols sedative effects. Direct comparison revealed distinct sex-dependent vulnerabilities: males showed reduced social interaction, whereas females exhibited numerically greater impairment in fear extinction and a significantly stronger ethanol preference. Baseline fear responses, total fluid intake, and sucrose consumption were unaffected. ConclusionPrenatal stress programs neurobehavioral trajectories in a sex-dependent manner, conferring vulnerability to anxiety-related behavior, cognitive disruption, and alcohol use. By directly comparing males and females within the same experimental design, this study provides one of the most integrated evaluations of sex-specific PNS outcomes to date and offers a robust framework for investigating the biological mechanisms underlying divergent pathways to stress-related psychopathology.

animal behavior and cognition↗

bioRxiv2025

A multifaceted approach for obstructive sleep apnea classification from ECG signal using deep learning

Obstructive sleep apnea (OSA) is a common sleep disorder associated with increased cardiovascular and neurocognitive risks. While polysomnography remains the clinical gold standard for diagnosis, it is costly and unsuitable for large-scale or real-time screening. Electrocardiogram (ECG) signals offer a non-invasive, low-cost alternative for sleep apnea detection. We present a holistic new framework for OSA detection and forecasting using ECG data on two datasets: PhysioNet Apnea-ECG datasets (healthy patients with apnea), and OSASUD dataset (patients in a stroke unit). Our framework integrates feature engineering methods rooted in dynamical systems theory and statistical analysis. These features are used across a range of models, from conventional machine learning algorithms to novel deep learning architectures. To improve generalization and personalization, we incorporate transfer learning in two ways: across datasets to adapt models trained on large cohorts to smaller clinical datasets, and at the patient level to personalize models using limited individual data, hence demonstrating the use of precision medicine.

bioinformatics↗

bioRxiv2025

Drug Repurposing Screen Identifies an HRI Activating Compound that Promotes Adaptive Mitochondrial Remodeling in MFN2-deficient Cells

Pathogenic variants in the mitochondrial outer membrane GTPase MFN2 cause the peripheral neuropathy Charcot-Marie-Tooth Type 2A (CMT2A). These mutations disrupt MFN2-dependent regulation of diverse aspects of mitochondrial biology including organelle morphology, motility, mitochondrial-endoplasmic reticulum (ER) contacts (MERCs), and respiratory chain activity. However, no therapies currently exist to mitigate the mitochondrial dysfunction linked to genetic deficiencies in MFN2. Herein, we performed a drug repurposing screen to identify compounds that selectively activate the integrated stress response (ISR) - the predominant stress-responsive signaling pathway responsible for regulating mitochondrial morphology and function. This screen identified the compounds parogrelil and MBX-2982 as potent and selective activators of the ISR through the OMA1-DELE1-HRI signaling axis. We show that treatment with these compounds promotes adaptive, ISR-dependent remodeling of mitochondrial morphology and protects mitochondria against genetic and chemical insults. Moreover, we show that pharmacologic ISR activation afforded by parogrelil restores mitochondrial tubular morphology, promotes mitochondrial motility, rescues MERCs, and enhances mitochondrial respiration in MFN2-deficient cells. These results demonstrate the potential for pharmacologic HRI activation as a viable strategy to mitigate mitochondrial dysfunction in CMT2A and other pathologies associated with MFN2 deficiency.

cell biology↗

bioRxiv2025

Thiazole substitution of a labile amide bond - a new option towards stable pantothenamide-mimics

The emergence and spread of artemisinin-resistant, malaria-causing P. falciparum provide the impetus for the development of novel antimalarials. Pantothenamides are potent inhibitors of malaria parasite proliferation, however their clinical use is hindered by pantetheinase-mediated degradation in human serum. Here we report the synthesis and biological activity of a series of pantothenamide-mimics in which the labile amide bond is replaced by a thiazole ring with various orientations. Out of 23 novel compounds generated and tested in the presence of pantetheinase, several display sub-micromolar antiplasmodial activity in vitro. A selection of compounds was studied in more detail and CoA biosynthesis and/or utilisation pathways were confirmed to be the target. Toxicity to human cells was not observed. Kinetic studies identified the selected compounds as substrates of the HsPanK3 enzyme, but with much lower affinity compared to that of the natural substrate pantothenate. The most potent thiazole-bearing antiplasmodial compound was found to bind to PfPanK with a 120-fold higher affinity compared to HsPanK, highlighting excellent selectivity, not only against the key first enzyme in the CoA biosynthesis pathway, but also at the whole-cell level. In conclusion, thiazole substitution of the labile amide bond represents a promising avenue for the development of antimalarial pantothenamide-mimics.

pharmacology and toxicology↗

bioRxiv2024

Ethograms reveal a fear conditioned visual cue to organize diverse behaviors in rats

Recognizing and responding to threat cues is essential to survival. In rats, freezing is the most common behavior measured. Previously we demonstrated a threat cue can organize diverse behaviors (Chu et al., 2024). However, the experimental design of Chu et al. (2024) was complex and the findings descriptive. Here, we gave female and male Long Evans rats simple paired or unpaired presentations of a light and foot shock (8 total) in a conditioned suppression setting, using a range of shock intensities (0.15, 0.25, 0.35 or 0.5 mA). We found that conditioned suppression was only observed at higher foot shock intensities (0.35 mA and 0.5 mA). We constructed comprehensive, temporal ethograms by scoring 22,272 frames of behavior for 12 mutually exclusive behavior categories in 200 ms intervals around cue presentation. A 0.5 mA and 0.35 mA shock-paired visual cue suppressed reward seeking, rearing and scaling, as well as light-directed rearing and light-directed scaling. The shock-paired visual paired cue further elicited locomotion and freezing. Linear discriminant analyses showed that ethogram data could accurately classify rats into paired and unpaired groups. Considering the complete ethogram data produced superior classification than considering subsets of behaviors. The results demonstrate diverse threat-elicited behaviors - in a simple Pavlovian fear conditioning design - containing sufficient information to distinguish the fear learning status of individual rats.

neuroscience↗

bioRxiv2024

Dissociable patterns of dopamine dynamics and causal contributions to stimulus-response behaviors across striatal subregions.

Rationale: Midbrain dopamine (DA) neurons project principally towards the striatum, serving as key regulators of movement, motivation, and cognition. Different striatal dopaminergic (DA) pathways may regulate different aspects of cognition. Objectives: We investigated how different striatal DA pathways contribute to a known function of the striatum, visuomotor conditional learning. Methods: Using fiber photometry, we recorded DA transients in these regions as mice learned the touchscreen Visuo-Motor Conditional Learning task. Results: DA transients in all regions dynamically tracked task events, but differed in the timing of peak responses and ramp-like activity preceding a choice, indicating region-specific temporal dynamics across learning. Manipulations of reward probability revealed DA transients in all regions during reward delivery and omission that are consistent with an interpretation in terms of reward prediction error. Thus, DA dynamics in all regions could indicate involvement in visuomotor conditional learning. Therefore, to determine whether nigrostriatal or mesolimbic DA is necessary for learning, we chemogenetically inhibited DA striatal afferents, revealing that only DLS-projecting nigrostriatal DA, and not NAc-projecting mesolimbic striatal DA, was necessary for learning the task. Conclusion: These findings demonstrate functional heterogeneity of aspects of striatal DA signaling, and selective causal roles in the learning of visuomotor conditional learning.

animal behavior and cognition↗

bioRxiv2024

Variants in tubule epithelial regulatory elements mediate most heritable differences in human kidney function

Kidney disease is highly heritable; however, the causal genetic variants, the cell types in which these variants function, and the molecular mechanisms underlying kidney disease remain largely unknown. To identify genetic loci affecting kidney function, we performed a GWAS using multiple kidney function biomarkers and identified 462 loci. To begin to investigate how these loci affect kidney function, we generated single-cell chromatin accessibility (scATAC-seq) maps of the human kidney and identified candidate cis-regulatory elements (cCREs) for kidney podocytes, tubule epithelial cells, and kidney endothelial, stromal, and immune cells. Kidney tubule epithelial cCREs explained 58% of kidney function SNP-heritability and kidney podocyte cCREs explained an additional 6.5% of SNP-heritability. In contrast, little kidney function heritability was explained by kidney endothelial, stromal, or immune cell-specific cCREs. Through functionally informed fine-mapping, we identified putative causal kidney function variants and their corresponding cCREs. Using kidney scATAC-seq data, we created a deep learning model (which we named ChromKid) to predict kidney cell type-specific chromatin accessibility from sequence. ChromKid and allele specific kidney scATAC-seq revealed that many fine-mapped kidney function variants locally change chromatin accessibility in tubule epithelial cells. Enhancer assays confirmed that fine-mapped kidney function variants alter tubule epithelial regulatory element function. To map the genes which these regulatory elements control, we used CRISPR interference (CRISPRi) to target these regulatory elements in tubule epithelial cells and assessed changes in gene expression. CRISPRi of enhancers harboring kidney function variants regulated NDRG1 and RBPMS expression. Thus, inherited differences in tubule epithelial NDRG1 and RBPMS expression may predispose to kidney disease in humans. We conclude that genetic variants affecting tubule epithelial regulatory element function account for most SNP-heritability of human kidney function. This work provides an experimental approach to identify the variants, regulatory elements, and genes involved in polygenic disease.

genetics↗

bioRxiv2024

The maternal microbiome modifies adverse effects of protein undernutrition on offspring neurobehavioral impairment in mice

Protein undernutrition is a global risk factor for impaired growth and neurobehavioral development in children. However, the critical periods, environmental interactions, and maternal versus neonatal influences on programming lasting behavioral abnormalities are poorly understood. In a mouse model of fetal growth restriction, limiting maternal protein intake particularly during pregnancy leads to cognitive and anxiety-like behavioral abnormalities in adult offspring, indicating a critical role for the gestational period. By cross-fostering newborn mice to dams previously exposed to either low protein or standard diet, we find that the adult behavioral impairments require diet-induced conditioning of both fetal development and maternal peripartum physiology, rather than either alone. This suggests that protein undernutrition during pregnancy directly disrupts fetal neurodevelopment and indirectly alters maternal state in ways that interact postnatally to precipitate behavioral deficits. Consistent with this, maternal protein restriction during pregnancy reduces the diversity of the maternal gut microbiome, modulates maternal serum metabolomic profiles, and yields widespread alterations in fetal brain transcriptomic and metabolomic profiles, including subsets of microbiome-dependent metabolites. Depletion of the maternal microbiome in protein-restricted dams further alters fetal brain gene expression and exacerbates neurocognitive behavior in adult offspring, suggesting that the maternal microbiome modifies the impact of gestational protein undernutrition on risk for neurobehavioral impairment in the offspring. To explore the potential for microbiome-targeted interventions, we find that maternal treatment with short chain fatty acids or a cocktail of 10 diet- and microbiome-dependent metabolites each yield differential effects on fetal development and/or postnatal behavior. Results from this study highlight impactful prenatal influences of maternal protein undernutrition on fetal neurodevelopment and adverse neurobehavioral trajectories in offspring, which are mitigated by microbiome-targeted interventions during pregnancy.

animal behavior and cognition↗

bioRxiv2023

Multi-modal skin atlas associates a multicellular immune-stromal community with altered cornification and T cell expansion in atopic dermatitis

In healthy skin, a cutaneous immune system maintains the balance between tolerance towards innocuous environmental antigens and immune responses against pathological agents. In atopic dermatitis (AD), barrier and immune dysfunction result in chronic tissue inflammation. Our understanding of the skin tissue ecosystem in AD remains incomplete with regard to the hallmarks of pathological barrier formation, and cellular state and clonal composition of disease-promoting cells. Here, we generated a multi-modal cell census of 310,691 cells spanning 86 cell subsets from whole skin tissue of 19 adult individuals, including non-lesional and lesional skin from 11 AD patients, and integrated it with 396,321 cells from four studies into a comprehensive human skin cell atlas in health and disease. Reconstruction of human keratinocyte differentiation from basal to cornified layers revealed a disrupted cornification trajectory in AD. This disrupted epithelial differentiation was associated with signals from a unique immune and stromal multicellular community comprised of MMP12+ dendritic cells (DCs), mature migratory DCs, cycling ILCs, NK cells, inflammatory CCL19+ IL4I1+ fibroblasts, and clonally expanded IL13+IL22+IL26+ T cells with overlapping type 2 and type 17 characteristics. Cell subsets within this immune and stromal multicellular community were connected by multiple inter-cellular positive feedback loops predicted to impact community assembly and maintenance. AD GWAS gene expression was enriched both in disrupted cornified keratinocytes and in cell subsets from the lesional immune and stromal multicellular community including IL13+IL22+IL26+ T cells and ILCs, suggesting that epithelial or immune dysfunction in the context of the observed cellular communication network can initiate and then converge towards AD. Our work highlights specific, disease-associated cell subsets and interactions as potential targets in progression and resolution of chronic inflammation.

molecular biology↗

bioRxiv2023

Optogenetic inhibition of the caudal substantia nigra inflates behavioral responding to uncertain threat and safety

Defensive responding is adaptive when it approximates current threat, but maladaptive when it exceeds current threat. Here we asked if the substantia nigra, a region consistently implicated in reward, is necessary to show appropriate levels of defensive responding in Pavlovian fear discrimination. Rats received bilateral transduction of the caudal substantia nigra with halorhodopsin or a control fluorophore, and bilateral ferrule implants. Rats then behaviorally discriminated cues predicting unique foot shock probabilities (danger, p=1; uncertainty, p=0.25; and safety, p=0). Green-light illumination (532 nm) during cue presentation inflated defensive responding of halorhodopsin rats - measured by suppression of reward seeking - to uncertainty and safety beyond control levels. Green-light illumination outside of cue presentation had no impact on halorhodopsin or control rat responding. The results reveal caudal substantia nigra cue activity is necessary to inhibit defensive responding to non-threatening and uncertain threat cues.

neuroscience↗

bioRxiv2022

A fear conditioned cue orchestrates a suite of behaviors

Pavlovian fear conditioning has been extensively used to study the behavioral and neural basis of defensive systems. In a typical procedure, a cue is paired with foot shock, and subsequent cue presentation elicits freezing, a behavior theoretically linked to predator detection. Studies have since shown a fear conditioned cue can elicit locomotion, a behavior that - in addition to jumping, and rearing - is theoretically linked to imminent or occurring predation. A criticism of studies observing fear conditioned cue-elicited locomotion is that responding is non-associative. We gave 24 rats (12 female) Pavlovian fear discrimination over a baseline of reward seeking. The within-subjects procedure had full controls for associative learning, consisting of three cues predicting unique foot shock probabilities: danger (p=1), uncertainty (p=0.25), and safety (p=0). TTL-triggered cameras captured 5 behavior frames/s prior to and during cue presentation. We scored 86,400 frames for nine discrete behaviors spanning reward, passive fear, and active fear. Temporal ethograms show that a fear conditioned cue elicits locomotion, jumping, and rearing that is maximal towards cue offset, when foot shock is imminent. A fear conditioned cue further suppresses reward-related behavior, and elicits freezing in a sex-specific manner. The differing temporal profiles and independent expression of these behaviors reveal a fear conditioned cue to orchestrate a rich and intricate suite of behaviors.

animal behavior and cognition↗

bioRxiv2021

AFP2 inhibits ABA responses during germination without ABI5 degradation but DWAs reduce desiccation tolerance

Overexpression of ABI5/ABF interacting proteins (AFPs) results in extreme ABA resistance of seeds and failure to acquire desiccation tolerance, at least in part through effects on chromatin modification. This study tests the hypothesis that the AFPs promote germination by also functioning as adapters for E3 ligases that ubiquitinate ABI5, leading to its degradation. Interactions between AFPs and two well-characterized classes of E3 ligases targeting ABI5, DWD HYPERSENSITIVE TO ABA (DWA)s and KEEP ON GOING (KEG), were analyzed by yeast two-hybrid, bimolecular fluorescence complementation, and genetic assays. Although the AFPs and E3 ligases showed weak direct interactions, loss of function for the E3 ligases did not impair ABA-resistance conferred by overexpression of the YFP-AFP2 fusion. Comparison of ABI5 and AFP2 levels in these lines showed that AFP2 accumulation increased during germination, but that ABI5 degradation followed germination, demonstrating that AFP2 controls ABA sensitivity during germination independently of ABI5 degradation. Surprisingly, AFP2 overexpression in the dwa1 dwa2 mutant background produced the unusual combination of extreme ABA resistance and desiccation tolerance, creating an opportunity to separate the underlying biochemical characteristics of ABA sensitivity and desiccation tolerance that we investigated by quantitative proteomics. Our analysis identified at least three-fold more differentially accumulated seed proteins than previous studies. Comparison of dry seed proteomes of the different genotypes allowed us to separate and refine the changes in protein accumulation patterns correlating with desiccation tolerance independently of ABA sensitivity, or vice versa, to a subset of cold-induced and defense stress-responsive proteins and signaling regulators. Summary SentenceExtreme ABA resistance conferred by overexpression of AFP2 is not mediated by interactions with E3 ligases, but the dwa background maintains desiccation tolerance despite ABA resistance.

plant biology↗

bioRxiv2021

Single-Cell Transcriptomic Analysis of Livers During NLRP3 Inflammasome Activation Reveals a Novel Immune Niche

The NOD-like receptor protein 3 (NLRP3) inflammasome is a central contributor to human acute and chronic liver disease, yet the molecular and cellular mechanisms by which its activation precipitates injury remain incompletely understood. Here, we present single cell transcriptomic profiling of livers from a global transgenic Tamoxifen-inducible constitutively-activated Nlrp3A350V mutant mouse, and we investigate the changes in parenchymal and non-parenchymal liver cell gene expression that accompany inflammation and fibrosis. Our results demonstrate that NLRP3 activation causes chronic extramedullary myelopoiesis marked by an increase in proliferating myeloid progenitors that differentiate into neutrophils, monocytes, and monocyte-derived macrophages, results that were corroborated by flow cytometry and histological staining. We observed prominent neutrophil infiltrates with increased Ly6gHI and Ly6gINT cells exhibiting transcriptomic signatures of granulopoiesis typically found in the bone marrow. This was accompanied by a marked increase in Ly6cHI monocytes differentiating into Cd11bHITim4HIClec4fHI macrophages that express proinflammatory transcriptional programs similar to macrophages of non-alcoholic steatohepatitis (NASH) models. NLRP3 activation also downregulated metabolic pathways in hepatocytes and shifted hepatic stellate cells towards an activated pro-fibrotic state based on expression of collagen and extracellular matrix (ECM) regulatory genes. These results, which highlight abundant neutrophils and extramedullary granulopoiesis define an inflamed and fibrotic hepatic single cell microenvironment, precipitated solely by NLRP3 activation. Clinically, our data support the notion that neutrophils and NLRP3 should be explored as therapeutic targets in NASH-like inflammation.

immunology↗

bioRxiv2021

Ezh2 as an epigenetic checkpoint regulator during monocyte differentiation: a potential target to improve cardiac repair after myocardial infarction

Epigenetic regulation of histone H3K27 methylation has recently emerged as a key step during M2-like macrophage polarization, essential for cardiac repair after Myocardial Infarction (MI). We demonstrate for the first-time that EZH2, responsible for H3K27 methylation, has an ectopic cytoplasmic localization during monocyte differentiation in M2 macrophages. Moreover, we show that pharmacological EZH2 inhibition, with GSK-343, enhances bivalent genes, expression to promote human monocyte repair functions. GSK-343 treatment accelerated cardiac inflammatory resolution preventing infarct expansion and subsequent cardiac dysfunction after MI in vivo. In conclusion, our study reveals that epigenetic modulation of cardiac-infiltrating immune cells may hold promise to limit adverse cardiac remodeling after MI.

physiology↗

bioRxiv2020

The structural basis of fatty acid elongation by the ELOVL elongases

Very long chain fatty acids (VLCFAs) are essential building blocks for synthesis of the ceramides and sphingolipids required for nerve, skin and retina function and 3-keto acyl-CoA synthases (ELOVL elongases) perform the first step in the FA elongation cycle. Although ELOVLs are implicated in common diseases including insulin resistance, hepatic steatosis and Parkinsons, their underlying molecular mechanisms are unknown. Here we report the structure of the human ELOVL7 elongase, which includes an inverted transmembrane barrel structure surrounding a 35 [A] long tunnel containing a covalently-attached product analogue. The structure reveals the substrate binding sites in the tunnel and an active site deep in the membrane including the canonical ELOVL HxxHH sequence. This indicates a ping-pong mechanism for catalysis, involving unexpected covalent histidine adducts. The unusual substrate-binding arrangement and chemistry suggest mechanisms for selective ELOVL inhibition, relevant for diseases where VLCFAs accumulate such as X-linked adrenoleukodystrophy.

biochemistry↗

bioRxiv2020

A new paradigm for Prelamin A proteolytic processing by ZMPSTE24: the upstream SY^LL cleavage occurs first and there is no CaaX processing by ZMPSTE24

Human ZMPSTE24, an integral membrane zinc metalloprotease, is required for conversion of prelamin A to mature lamin A, a component of the nuclear lamina and failure of this processing causes premature ageing disorders. ZMPSTE24 has also been implicated in both type 2 diabetes mellitus and in viral-host response mechanisms, but to date its only confirmed substrate is the precursor for lamin A. Prelamin A is thought to undergo four C-terminal post-translational modifications in the following order: farnesylation, SIM tripeptide cleavage, carboxymethylation and upstream "SY^LL" cleavage. Here we present evidence that the sequence of events does not follow the accepted dogma. We assessed cleavage of long human prelamin A sequence peptides by purified human ZMPSTE24 combined with FRET and mass spectrometry to detect products. Surprisingly, we found that the "SY^LL" cleavage occurs before and independent of the C-terminal CSIM modifications. We also found that ZMPSTE24 does not perform the predicted C^SIM tripeptide cleavage, but rather it removes an IM dipeptide. ZMPSTE24 can perform a tripeptide cleavage with a canonical CaaX box (C: cysteine; a: aliphatic; X: any residue), but the C-terminus of prelamin A is not a true CaaX sequence. Regardless of the C-terminal modifications of prelamin A, ZMPSTE24 can perform upstream SY^LL cleavage, thus removing the unwanted farnesylated C-terminus. Therefore, it is failure of SY^LL cleavage, not the C-terminal processing that is the likely cause of progeroid disorders.

biochemistry↗

bioRxiv2020

Selective Plane Illumination Microscopy and Computing Reveal Differential Obliteration of Retinal Vascular Plexuses

Murine models of visual impairment provide micro-vascular insights into the 3-D network disarray in retinopathy. Current imaging and analysis tend to be confined to the 2-D retinal vasculature. We hereby integrated selective plane illumination imaging or known as light-sheet fluorescence microscopy (LSFM) with dual-illumination, followed by computational analyses, to reveal the topological network of vertical sprouts bridging the primary and secondary plexuses in a postnatal mouse model of oxygen-induced retinopathy (OIR). We revealed a preferential obliteration of the secondary plexus and bridging vessels despite a relatively unscathed primary plexus. We compared the local versus global vascular connectivity using clustering coefficients and Euler numbers, respectively. The global vascular connectivity in hyperoxia-exposed retinas was significantly reduced (p < 0.05, n = 5 vs. normoxia), whereas the local connectivity was preserved (p > 0.05, n = 5 vs. normoxia). We further applied principal component analysis (PCA) to automatically segment the vertical sprouts, corroborating the preferential obliteration of the interconnection between vertical sprouts and secondary plexuses that were accompanied with impaired vascular branching and connectivity, and reduced vessel volumes and lengths (p < 0.05, n=5 vs. normoxia). Thus, integration of 3-D selective plane illumination with computational analyses allows for early detection of global and spatially-specific vaso-obliteration, but preserved local reticular structure in response to hyperoxia-induced retinopathy.

bioengineering↗