SUMMARY Complement dysregulation is a feature of many retinal diseases, yet mechanistic understanding at the cellular level is limited. Given this knowledge gap about which retinal cells express complement, we performed single-cell RNA sequencing on ~92,000 mouse retinal cells and validated our results in five major purified retinal cell types. We found evidence for a distributed cell-type-specific complement expression across 11 cell types. Notably, Müller cells are the major contributor of complement activators c1s, c3, c4, and cfb. Retinal pigment epithelium (RPE) mainly expresses cfh and the terminal complement components, whereas cfi and cfp transcripts are most abundant in neurons. Aging enhances c1s, cfb, cfp, and cfi expression, while cfh expression decreases. Transient retinal ischemia increases complement expression in microglia, Müller cells, and RPE. In summary, we report a unique complement expression signature for murine retinal cell types suggesting a well-orchestrated regulation of local complement expression in the retinal microenvironment.

Background Radiofrequency (RF) ablation to treat atrial arrhythmia is limited by an inability to reliably assess lesion durability and transmurality. Objective Determine feasibility of photoacoustic characterization of myocardial ablation lesions in vitro. In this study, we investigate the feasibility of combined ultrasound (US) and spectroscopic photoacoustic (sPA) imaging to visualize RF ablation lesions in 3-D based on unique differences in the optical absorption spectra between normal and ablated myocardial tissue. Methods Tissue samples were excised from the ventricles of fresh porcine hearts. Lesions were generated using an RF catheter ablation system using 20 - 30 W of power applied for 40 - 60 s. Ablated samples were imaged in the NIR regime (740-780 nm) using a combined PA/US imaging system. Measured PA spectra were correlated to the absorption spectra of deoxy-hemoglobin and ablated tissue to produce a tissue characterization map (TCM) identifying 3-D lesion location and extent. Tissue samples were stained and photographed for gross pathology. TCM and gross pathology images were co-registered to assess TCM accuracy. Results The TCM reliably characterized ablated and non-ablated tissue up to depths of 3 mm. The TCM also assessed lesion position and extent with sub-millimeter accuracy in multiple dimensions. Segmented TCMs achieved greater than 69% agreement with gross pathology. Conclusion These results suggest that sPA imaging has the potential to accurately assess RF ablation lesion size and position with sub-millimeter precision and may be well suited to guide trans-catheter RF atrial ablation in clinical practice.

The intent of the current study was to investigate the therapeutic contribution of MSCs to vascular regeneration and functional recovery of ischemic tissue. We used a rodent hind limb ischemia model and intramuscularly delivered MSCs within a PEGylated fibrin gel matrix. Within this model, we demonstrated that MSC therapy, when delivered in PEGylated fibrin, results in significantly higher mature blood vessel formation, which allows for greater functional recovery of skeletal muscle tissue as assessed using force production measurements. We observed initial signs of vascular repair at early time points when MSCs were delivered without PEGylated fibrin, but this did not persist or lead to recovery of the tissue in the long-term. Furthermore, animals which were treated with PEGylated fibrin alone exhibited a greater number of mature blood vessels, but they did not arterialize and did not show improvements in force production. These results demonstrate that revascularization of ischemic tissue may be a necessary but not sufficient step to complete functional repair of the injured tissue. This work has implications on stem cell therapies for ischemic diseases and also potentially on how such therapies are evaluated.

Age‐related macular degeneration (AMD) is a blinding eye disease with no unifying theme for its etiology. We used single-cell RNA sequencing to analyze the transcriptomes of ~ 93,000 cells from the macula and peripheral retina from two adult human donors and bulk RNA sequencing from fifteen adult human donors with and without AMD. Analysis of our single-cell data identified 267 cell-type-specific genes. Comparison of macula and peripheral retinal regions found no cell-type differences but did identify 50 differentially expressed genes (DEGs) with about 1/3 expressed in cones. Integration of our single-cell data with bulk RNA sequencing data from normal and AMD donors showed compositional changes more pronounced in macula in rods, microglia, endothelium, Müller glia, and astrocytes in the transition from normal to advanced AMD. KEGG pathway analysis of our normal vs. advanced AMD eyes identified enrichment in complement and coagulation pathways, antigen presentation, tissue remodeling, and signaling pathways including PI3K-Akt, NOD-like, Toll-like, and Rap1. These results showcase the use of single-cell RNA sequencing to infer cell-type compositional and cell-type-specific gene expression changes in intact bulk tissue and provide a foundation for investigating molecular mechanisms of retinal disease that lead to new therapeutic targets.