Fly-scan high-throughput coded ptychographic microscopy via active micro-vibration and rolling-shutter distortion correction.

Recent advancements in ptychography have demonstrated the potential of coded ptychography (CP) for high-resolution optical imaging in a lensless configuration. However, CP suffers imaging throughput limitations due to scanning inefficiencies. To address this, we propose what we believe is a novel 'fly-scan' scanning strategy utilizing two eccentric rotating mass (ERM) vibration motors for high-throughput coded ptychographic microscopy. The intrinsic continuity of the 'fly-scan' technique effectively eliminates the scanning overhead typically encountered during data acquisition. Additionally, its randomized scanning trajectory considerably reduces periodic artifacts in image reconstruction. We also developed what we believe to be a novel rolling-shutter distortion correction algorithm to fix the rolling-shutter effects. We built up a low-cost, DIY-made prototype platform and validated our approach with various samples including a resolution target, a quantitative phase target, a thick potato sample and biospecimens. The reported platform may offer a cost-effective and turnkey solution for high-throughput bio-imaging.

DIRECT: Digital Microfluidics for Isolation-Free Shared Library Construction of Single-Cell DNA Methylome and Transcriptome.

Simultaneous profiling of DNA methylation and gene expression within single cells is a powerful technology to dissect complex gene regulatory network of cells. However, existing methods are based on picking a single-cell in a tube and split single-cell lysate into two parts for transcriptome and methylome library construction, respectively, which is costly and cumbersome. Here, DIRECT is proposed, a digital microfluidics-based method for high-efficiency single-cell isolation and simultaneous analysis of the methylome and transcriptome in a single library construction. The accuracy of DIRECT is demonstrated in comparison with bulk and single-omics data, and the high CpG site coverage of DIRECT allows for precise analysis of copy number variation information, enabling expansion of single cell analysis from two- to three-omics. By applying DIRECT to monitor the dynamics of mouse embryonic stem cell differentiation, the relationship between DNA methylation and changes in gene expression during differentiation is revealed. DIRECT enables accurate, robust, and reproducible single-cell DNA methylation and gene expression co-analysis in a more cost-effective, simpler library preparation and automated manner, broadening the application scenarios of single-cell multi-omics analysis and revealing a more comprehensive and fine-grained map of cellular regulatory landscapes.

Structured modulation multi-height microscopy for high-resolution imaging.

Conventional multi-height microscopy techniques introduce different object-to-detector distances to obtain multiple measurements for phase retrieval. However, surpassing the diffraction limit imposed by the numerical aperture (NA) of the objective lens remains a challenging task. Here, we report a novel structured modulation multi-height microscopy technique for quantitative high-resolution imaging. In our platform, a thin diffuser is placed in between the sample and the objective lens. By translating the diffuser to different axial positions, a sequence of modulated intensity images is captured for reconstruction. The otherwise inaccessible high-resolution object information can thus be encoded into the optical system for detection. In the construction process, we report a ptychographic phase retrieval algorithm to recover the existing wavefront of the complex object. We validate our approach using a resolution target, a phase target, and various biological samples. We demonstrate a ∼4-fold resolution gain over the diffraction limit. We also demonstrate our approach to achieve a 6.5 mm by 4.3 mm field of view and a half-pitch resolution of 1.2 µm. The reported methodology provides a portable, turnkey solution for quantitative high-resolution imaging with potential applications in disease diagnosis, sample screening, and other fields.

Digital-scRRBS: A Cost-Effective, Highly Sensitive, and Automated Single-Cell Methylome Analysis Platform via Digital Microfluidics.

Single-cell DNA methylation sequencing is highly effective for identifying cell subpopulations and constructing epigenetic regulatory networks. Existing methylome analyses require extensive starting materials and are costly, complex, and susceptible to contamination, thereby impeding the development of single-cell methylome technology. In this work, we report digital microfluidics-based single-cell reduced representation bisulfite sequencing (digital-scRRBS), the first microfluidics-based single-cell methylome library construction platform, which is an automatic, effective, reproducible, and reagent-efficient technique to dissect the single-cell methylome. Using our digital microfluidic chip, we isolated single cells in 15 s and successfully constructed single-cell methylation sequencing libraries with a unique genome mapping rate of up to 53.6%, covering up to 2.26 million CpG sites. Digital-scRRBS demonstrates a high capacity for distinguishing cell identity and tracking DNA methylation during drug administration. Digital-scRRBS expands the applicability of single-cell methylation methods as a versatile tool for epigenetic analysis of rare cells and populations with high levels of heterogeneity.

PAI-1 genetic polymorphisms influence septic patients' outcomes by regulating neutrophil activity.

BACKGROUND: Plasminogen activator inhibitor-1 (PAI-1) plays an important role in the pathophysiology of sepsis, but the exact mechanism remains debatable. In this study, we investigated the associations among the serum levels of PAI-1, the incidence of 4G/5G promoter PAI-1 gene polymorphisms, immunological indicators, and clinical outcomes in septic patients. METHODS: A total of 181 patients aged 18-80 years with sepsis between November 2016 and August 2018 in the intensive care unit in the Xinhua Hospital were recruited in this retrospective study, with 28-day mortality as the primary outcome. The initial serum level of PAI-1 and the presence of rs1799768 single nucleotide polymorphisms (SNPs) were examined. Univariate logistic regression and multivariate analyses were performed to determine the factors associated with different genotypes of PAI-1, serum level of PAI-1, and 28-day mortality. RESULTS: The logistic analysis suggested that a high serum level of PAI-1 was associated with the rs1799768 SNP of PAI-1 (4G/4G and 4G/5G) (Odds ratio [OR]: 2.49; 95% confidence interval [CI]: 1.09, 5.68). Furthermore, a high serum level of PAI-1 strongly influenced 28-day mortality (OR 3.36; 95% CI 1.51, 7.49). The expression and activation of neutrophils (OR 0.96; 95% CI 0.93, 0.99), as well as the changes in the expression patterns of cytokines and chemokine-associated neutrophils (OR: 1.00; 95% CI: 1.00, 1.00), were both regulated by the genotype of PAI-1. CONCLUSIONS: Genetic polymorphisms of PAI-1 can influence the serum levels of PAI-1, which might contribute to mortality by affecting neutrophil activity. Thus, patients with severe sepsis might clinically benefit from enhanced neutrophil clearance and the resolution of inflammation via the regulation of PAI-1 expression and activity.

Plasminogen Activator Inhibitor-1 Potentiates Neutrophil Infiltration and Tissue Injury in Colitis.

The mechanism underlying inflammatory bowel disease (IBD) remains unclear. We aimed to identify early diagnostic biomarkers and understand their roles in the pathogenesis of IBD. Methods: We identified plasminogen activator inhibitor-1 (PAI-1) as a potential key gene that is upregulated in IBD based on published transcriptomic datasets. To further determine the role of PAI-1 in disease pathogenesis, we induced colitis in wild-type (WT) and PAI-1 knockout (KO) mice by administering dextran sulfate sodium (DSS). We used an RNA array of genes and 16S rRNA sequencing of the microbiome to analyze PAI-1 function. The colon and serum PAI-1 levels in humans were further evaluated for their diagnostic value. Results: PAI-1 expression was significantly increased in patients and DSS-induced WT mice but reduced in PAI-1 KO mice. These changes were associated with significantly decreased neutrophil infiltration in colonic tissues. The RNA array revealed that the CXC chemokines CXCL1 and CXCL5 and their common receptor CXCR2 were among the most significantly different genes between the PAI-1 KO mice with DSS-induced colitis and the WT mice. Mechanistically, PAI-1 deficiency led to blunted activation of the NF-κB pathway in the colon epithelium. The gut microbiome was altered in the PAI-1 KO mice, which showed enriched abundances of short-chain fatty acid-producing genera and diminished abundances of pathogenic genera. Receiver operating characteristic (ROC) curve analysis revealed the diagnostic value of PAI-1. Conclusions: Our data suggest a previously unknown function of PAI-1 inducing neutrophil-mediated chemokine expression by activating the NF-κB pathway and affecting the function of the gut microbiome. PAI-1 could be a potential diagnostic biomarker and a therapeutic target in IBD.

Precipitation regulates the responses of xylem phenology of two dominant tree species to temperature in arid and semi-arid forest of the southern Altai Mountains.

Arid and semi-arid forests are important carbon sinks, with implications for the global carbon balance. However, the impacts of climate warming on the growth of arid and semi-arid forest tree species and ecosystem carbon sink dynamics remain uncertain because the effects of the complex interactions between precipitation and temperature on xylem phenology are not clearly understood. Here, we monitored xylem formation over two years in two dominant tree species (Siberian larch, Larix sibirica Ledeb.; Siberian spruce, Picea obovata Ledeb.) along the arid and semi-arid southern Altai Mountains of Central Asia. We determined that temperature interaction with precipitation plays a key role in regulating xylem phenology of these two species, with differences between species. Under rising mean annual temperatures, the growth of L. sibirica advanced as the onset of xylem formation was not limited by early season water availability. However, the earlier cessation of cell enlargement, likely due to legacy effects, compensated for such advancement. In contrast, water stress constrained the advancement of xylem formation under rising temperatures in P. obovata. Nevertheless, water stress was seemingly relieved later in the growing season and consequently did not lead to the earlier cessation of xylem formation. Our results demonstrate that precipitation drives species-specific response to rising temperatures and thus is a key driver of growing season length and carbon sink dynamics in arid and semi-arid forests under climate warming. Integrating the effects of temperature and precipitation on xylem phenology in climate models may improve estimates of climate-carbon feedback in arid and semi-arid forests under future warming scenarios.

Spatial- and Fourier-domain ptychography for high-throughput bio-imaging.

First envisioned for determining crystalline structures, ptychography has become a useful imaging tool for microscopists. However, ptychography remains underused by biomedical researchers due to its limited resolution and throughput in the visible light regime. Recent developments of spatial- and Fourier-domain ptychography have successfully addressed these issues and now offer the potential for high-resolution, high-throughput optical imaging with minimal hardware modifications to existing microscopy setups, often providing an excellent trade-off between resolution and field of view inherent to conventional imaging systems, giving biomedical researchers the best of both worlds. Here, we provide extensive information to enable the implementation of ptychography by biomedical researchers in the visible light regime. We first discuss the intrinsic connections between spatial-domain coded ptychography and Fourier ptychography. A step-by-step guide then provides the user instructions for developing both systems with practical examples. In the spatial-domain implementation, we explain how a large-scale, high-performance blood-cell lens can be made at negligible expense. In the Fourier-domain implementation, we explain how adding a low-cost light source to a regular microscope can improve the resolution beyond the limit of the objective lens. The turnkey operation of these setups is suitable for use by professional research laboratories, as well as citizen scientists. Users with basic experience in optics and programming can build the setups within a week. The do-it-yourself nature of the setups also allows these procedures to be implemented in laboratory courses related to Fourier optics, biomedical instrumentation, digital image processing, robotics and capstone projects.

Optical ptychography for biomedical imaging: recent progress and future directions [Invited].

Ptychography is an enabling microscopy technique for both fundamental and applied sciences. In the past decade, it has become an indispensable imaging tool in most X-ray synchrotrons and national laboratories worldwide. However, ptychography's limited resolution and throughput in the visible light regime have prevented its wide adoption in biomedical research. Recent developments in this technique have resolved these issues and offer turnkey solutions for high-throughput optical imaging with minimum hardware modifications. The demonstrated imaging throughput is now greater than that of a high-end whole slide scanner. In this review, we discuss the basic principle of ptychography and summarize the main milestones of its development. Different ptychographic implementations are categorized into four groups based on their lensless/lens-based configurations and coded-illumination/coded-detection operations. We also highlight the related biomedical applications, including digital pathology, drug screening, urinalysis, blood analysis, cytometric analysis, rare cell screening, cell culture monitoring, cell and tissue imaging in 2D and 3D, polarimetric analysis, among others. Ptychography for high-throughput optical imaging, currently in its early stages, will continue to improve in performance and expand in its applications. We conclude this review article by pointing out several directions for its future development.

Remote referencing strategy for high-resolution coded ptychographic imaging.

The applications of conventional ptychography are limited by its relatively low resolution and throughput in the visible light regime. The new development of coded ptychography (CP) has addressed these issues and achieved the highest numerical aperture for large-area optical imaging in a lensless configuration. A high-quality reconstruction of CP relies on precise tracking of the coded sensor's positional shifts. The coded layer on the sensor, however, prevents the use of cross correlation analysis for motion tracking. Here we derive and analyze the motion tracking model of CP. A novel, to the best of our knowledge, remote referencing scheme and its subsequent refinement pipeline are developed for blind image acquisition. By using this approach, we can suppress the correlation peak caused by the coded surface and recover the positional shifts with deep sub-pixel accuracy. In contrast with common positional refinement methods, the reported approach can be disentangled from the iterative phase retrieval process and is computationally efficient. It allows blind image acquisition without motion feedback from the scanning process. It also provides a robust and reliable solution for implementing ptychography with high imaging throughput. We validate this approach by performing high-resolution whole slide imaging of bio-specimens.

Depth-multiplexed ptychographic microscopy for high-throughput imaging of stacked bio-specimens on a chip.

Imaging a large number of bio-specimens at high speed is essential for many biomedical applications. The common strategy is to place specimens at different lateral positions and image them sequentially. Here we report a new on-chip imaging strategy, termed depth-multiplexed ptychographic microscopy (DPM), for parallel imaging and sensing at high speed. Different from the common strategy, DPM stacks multiple specimens in the axial direction and images the entire z-stack all at once. In our prototype platform, we modify a low-cost car mirror for programmable steering of the incident laser beam. A blood-coated image sensor is then placed underneath the stacked sample for acquiring the resulting diffraction patterns. With the captured images, we perform blind recovery of the incident beam angle and model different layers of the stacked sample as different coded surfaces for object reconstruction. For in vitro experiment, we demonstrate time-lapse cell culture monitoring by imaging 3 stacked microfluidic channels on the coded sensor. For high-throughput cytometric analysis, we image 5 stacked brain sections with a 205-mm2 field of view in ∼50 s. Cytometric analysis is also performed to quantify the cellular proliferation biomarkers on the slides. The DPM approach adds a new degree of freedom for data multiplexing in microscopy, enabling parallel imaging of multiple specimens using a single detector. The demonstrated 6-mm depth of field is among the longest ones in microscopy imaging. The novel depth-multiplexed configuration also complements the miniaturization provided by microfluidics devices, offering a solution for on-chip sensing and imaging with efficient sample handling.

Addition of Psathyrostachys huashanica HMW glutenin subunit expresses positive contribution to protein polymerization and gluten microstructure of receptor wheat.

Psathyrostachys huashanica Keng (2n = 2x = 14, NsNs) is considered as a valuable wild germplasm for wheat improvement on account of its numerous outstanding traits. During this study, 7182-1Ns with higher quality was screened out, a series of experiments were conducted to clarify the reasons of quality improvement. The results indicated 7182-1Ns was carried a novel high-molecular-weight glutenin subunit (HMW-GS) from P. huashanica, designated as P. huashanica' subunit in wheat (HS), which changed the HMW-GS compositions, increased the proportion of glutenins in wheat gluten protein, accelerated the accumulation speed of unextractable polymeric protein (UPP) during grain development stage accelerated, and a denser microstructure of the gluten network was formed in the dough. Therefore, the current research provides important reference on effectively utilize 7182-1Ns as an intermediate germplasm for quality breeding improvement.

miRNAs as Biomarkers and Possible Therapeutic Strategies in Synovial Sarcoma.

Synovial sarcoma (SS) is an epithelial-differentiated malignant stromal tumor that has the highest incidence in young people and can occur almost anywhere in the body. Many noncoding RNAs are involved in the occurrence, development, or pathogenesis of SS. In particular, the role of MicroRNAs (miRNAs) in SS is receiving increasing attention. MiRNA is a noncoding RNA abundant in cells and extracellular serums. Increasing evidence suggests that miRNA has played a significant role in the incidence and development of tumors in recent years, including sarcomas. Previous studies show that various sarcomas have their unique miRNA expression patterns and that various miRNA expression profiles can illustrate the classes of miRNAs that may elicit cancer-relevant activities in specific sarcoma subtypes. Furthermore, SS has been reported to have the most number of differentially expressed miRNAs, which indicated that miRNA is linked to SS. In fact, according to many publications, miRNAs have been shown to have a role in the development and appearance of SS in recent years, according to many publications. Since many studies showing that various miRNAs have a role in the development and appearance of SS in recent years have not been systematically summarized, we summarize the recent studies on the relationship between miRNA and SS in this review. For example, miR-494 promotes the development of SS via modulating cytokine gene expression. The role of miR-494-3p as a tumor suppressor is most likely linked to the CXCR4 (C-X-C chemokine receptor 4) regulator, although the exact mechanism is unknown. Our review aims to reveal in detail the potential biological value and clinical significance of miRNAs for SS and the potential clinical value brought by the association between SS and miRNAs.

Environmental factors have a major effect in shaping the gene expression of Siberian larch in the Altai Mountains of China.

The differentiation of gene expression is an important link between genotype and phenotype and has important contributions to species adaptation and ecosystem evolution. As a major component of the world's forests, boreal forests play an important role in regulating the global climate, and the phenology of tree species has been and is undergoing changes during global warming. Here, to understand the impact of global warming on gene expression in boreal forest species, we used PacBio and Illumina sequencing methods to study the transcriptome of natural populations of Siberian larch (Larix sibirica Ledeb.) from the Altai Mountains in Xinjiang, China. We found that populations in this area had low genetic differentiation, but individuals were genetically clustered together when they had close geographic distance. Environmental factors, especially temperature, dominated differential gene expression of Siberian larch, while the contribution of genetic variation is relatively small. We speculate that Siberian larch adapts to changes in temperature and precipitation by altering its own gene expression. These results not only predict the tolerance of boreal forests to higher temperatures in the future, but also inform forest management strategies under global climate change.

Deep distributed optimization for blind diffuser-modulation ptychography.

Blind diffuser-modulation ptychography has emerged as a low-cost technique for micro-nano holographic imaging, which enables breaking the resolution limit of optical systems. However, the existing reconstruction method requires thousands of measurements to recover object and diffuser profile simultaneously, which makes the data acquisition time-consuming and cumbersome. In this Letter, we report a novel, to the best of our knowledge, blind ptychography technique with deep distributed optimization, termed BPD2O. It decomposes the complicated optimization task into subproblems, then introduces extended ptychographical iterative engine and enhanced network solver to optimize each in a distributed strategy. In this way, BPD2O combines the advantages of both model-driven and data-driven strategies, realizing high-fidelity robust ptychography imaging. Extensive experiments validate that BPD2O can realize better resolution and lead to a reduction of more than one order of magnitude in the number of measurements.

Multi-omic Profiling Reveals that Intra-abdominal-Hypertension-Induced Intestinal Damage Can Be Prevented by Microbiome and Metabolic Modulations with 5-Hydroxyindoleacetic Acid as a Diagnostic Marker.

Emerging evidence shows that modulation of the microbiome can suppress intra-abdominal hypertension (IAH)-induced intestinal barrier damage through the regulation of amino acid (AA) biosynthesis. Here, we investigated the protective effects of orally gavaged Lactobacillus acidophilus L-92 (L92) and a mixture of AA in rats with induced IAH. The results showed that both L92 and AA pretreatments effectively mitigated IAH-induced intestinal damage. Interestingly, L92 but not AA prevented metagenomic changes induced by IAH. Bacteroides fragilis, Bacteroides eggerthii, Bacteroides ovatus, Faecalibacterium prausnitzii, Prevotella, and extensively altered functional pathways were associated with L92-mediated host protection. Metabolomic profiling revealed that tryptophan metabolism was involved in both L92- and AA-mediated gut protection. The tryptophan metabolite 5-hydroxyindoleacetic acid (5-HIAA) is a sensitive biomarker for IAH in rats and patients with either gut-derived sepsis (n = 41) or all-source sepsis (n = 293). In conclusion, we show that microbiome and metabolic modulations can effectively prevent IAH-induced intestinal damage and that 5-HIAA is a potential metabolic marker for IAH and sepsis. IMPORTANCE Gut protection through modulation of the microbiome for critically ill patients has been gaining much attention recently. Intra-abdominal hypertension (IAH) is a prevailing clinical feature of acute gastrointestinal injuries in critically ill patients, characterized by nonspecific intestinal barrier damage. Prolonged IAH can induce or aggravate the development of sepsis and multiorgan dysfunctions. Therefore, the prevention of IAH-induced damage in rats through microbiome and metabolic interventions by commercially available L92 and AA treatments and the identification of 5-HIAA as an important marker for IAH/sepsis have important clinical implications for the treatment and early diagnosis of critically ill patients.

Low-Temperature Vapor-Phase Anion-Exchange Strategy for Wide-Bandgap Double-Perovskite Cs2AgBiCl6 Films toward Weak Ultraviolet Light Imaging.

Low-temperature synthesis of high-quality, high-stability, wide-bandgap perovskite films by solution methods is still challenging. Herein, large-scale wide-bandgap Cs2AgBiCl6 (CABC) double perovskite films are synthesized by a vapor-phase anion-exchange strategy. By dedicatedly designing an ultrathin TiO2 modification layer between the substrate and double perovskites, high-quality heterojunctions with matched energy band alignment are formed, contributing to a remarkably enhanced ON/OFF ratio of 2.4 × 104 (86 times) and a responsivity of 16 mA W-1 (12 times). Additionally, the ultraviolet photodetectors (UV PDs) exhibit an excellent UV detection limit of 1.18 µW cm-2 (20 nW), a broad linear dynamic range of 146 dB, and a high specific detectivity of 2.06 × 1011 Jones, as well as long-term stability. Finally, we further demonstrate a weak UV imaging system using CABC UV PDs as imaging sensors. The system is capable of imaging weak UV signals as low as 2.94 µW cm-2 (50 nW). Our results provide a feasible approach for low-temperature fabrication of wide-bandgap perovskite UV PDs and explore the promising application for weak UV detection and imaging.

High-throughput digital pathology via a handheld, multiplexed, and AI-powered ptychographic whole slide scanner.

The recent advent of whole slide imaging (WSI) systems has moved digital pathology closer to diagnostic applications and clinical practices. Integrating WSI with machine learning promises the growth of this field in upcoming years. Here we report the design and implementation of a handheld, colour-multiplexed, and AI-powered ptychographic whole slide scanner for digital pathology applications. This handheld scanner is built using low-cost and off-the-shelf components, including red, green, and blue laser diodes for sample illumination, a modified stage for programmable sample positioning, and a synchronized image sensor pair for data acquisition. We smear a monolayer of goat blood cells on the main sensor for high-resolution lensless coded ptychographic imaging. The synchronized secondary sensor acts as a non-contact encoder for precisely tracking the absolute object position for ptychographic reconstruction. For WSI, we introduce a new phase-contrast-based focus metric for post-acquisition autofocusing of both stained and unstained specimens. We show that the scanner can resolve the 388-nm linewidth on the resolution target and acquire gigapixel images with a 14 mm × 11 mm area in ∼70 seconds. The imaging performance is validated with regular stained pathology slides, unstained thyroid smears, and malaria-infected blood smears. The deep neural network developed in this study further enables high-throughput cytometric analysis using the recovered complex amplitude. The reported do-it-yourself scanner offers a portable solution to transform the high-end WSI system into one that can be made widely available at a low cost. The capability of high-throughput quantitative phase imaging may also find applications in rapid on-site evaluations.

Blood-Coated Sensor for High-Throughput Ptychographic Cytometry on a Blu-ray Disc.

The Blu-ray drive is an engineering masterpiece that integrates disc rotation, pickup head translation, and three lasers in a compact and portable format. Here, we integrate a blood-coated image sensor with a modified Blu-ray drive for high-throughput cytometric analysis of various biospecimens. In this device, samples are mounted on the rotating Blu-ray disc and illuminated by the built-in lasers from the pickup head. The resulting coherent diffraction patterns are then recorded by the blood-coated image sensor. The rich spatial features of the blood-cell monolayer help down-modulate the object information for sensor detection, thus forming a high-resolution computational biolens with a theoretically unlimited field of view. With the acquired data, we develop a lensless coherent diffraction imaging modality termed rotational ptychography for image reconstruction. We show that our device can resolve the 435 nm line width on the resolution target and has a field of view only limited by the size of the Blu-ray disc. To demonstrate its applications, we perform high-throughput urinalysis by locating disease-related calcium oxalate crystals over the entire microscope slide. We also quantify different types of cells on a blood smear with an acquisition speed of ∼10,000 cells per second. For in vitro experiments, we monitor live bacterial cultures over the entire Petri dish with single-cell resolution. Using biological cells as a computational lens could enable new intriguing imaging devices for point-of-care diagnostics. Modifying a Blu-ray drive with the blood-coated sensor further allows the spread of high-throughput optical microscopy from well-equipped laboratories to citizen scientists worldwide.

CD47 Blockade Accelerates Blood Clearance and Alleviates Early Brain Injury After Experimental Subarachnoid Hemorrhage.

Aims: Subarachnoid hemorrhage (SAH) is a devastating stroke subtype. Following SAH, erythrocyte lysis contributes to cell death and brain injuries. Blockage of the anti-phagocytic receptor Cluster of Differentiation 47 (CD47) enhances phagocyte clearance of erythrocytes, though it has not been well-studied post-SAH. The current study aims to determine whether anti-CD47 treatment can enhance blood clearance after experimental SAH. Methods: The prechiasmatic blood injection model of SAH was used in mice. Mice were either treated with the CD47-blocking antibody or IgG as control. The effect of the anti-CD47 antibody on blood clearance and neurological function following SAH was determined. Neuroinflammation and neuronal injury were compared between the treatment and control samples on day 1 and day 7 after SAH using flow cytometry, immunofluorescence, Fluoro-Jade C, and Nissl staining, RT-PCR, and Western blot analysis. Results: CD47-blocking antibody sped-up blood clearance after SAH, and resulted in less neuronal injury and neurological deficits than control samples. Microglia played a role in the anti-CD47 blockade. Following SAH Following SAH, CD47 antibody-treated mice had less neuroinflammation and lower levels of apoptosis compared to controls and both one and 7 days. Conclusions: CD47 antibody treatment has a neuroprotective effect following SAH, by increasing blood clearance rate and reducing brain injury. These findings suggest CD47 antibody treatment may improve SAH patient outcomes.