CD163+ macrophages monitor enhanced permeability at the blood-dorsal root ganglion barrier.

In dorsal root ganglia (DRG), macrophages reside close to sensory neurons and have largely been explored in the context of pain, nerve injury, and repair. However, we discovered that most DRG macrophages interact with and monitor the vasculature by sampling macromolecules from the blood. Characterization of the DRG vasculature revealed a specialized endothelial bed that transformed in molecular, structural, and permeability properties along the arteriovenous axis and was covered by macrophage-interacting pericytes and fibroblasts. Macrophage phagocytosis spatially aligned with peak endothelial permeability, a process regulated by enhanced caveolar transcytosis in endothelial cells. Profiling the DRG immune landscape revealed two subsets of perivascular macrophages with distinct transcriptome, turnover, and function. CD163+ macrophages self-maintained locally, specifically participated in vasculature monitoring, displayed distinct responses during peripheral inflammation, and were conserved in mouse and man. Our work provides a molecular explanation for the permeability of the blood-DRG barrier and identifies an unappreciated role of macrophages as integral components of the DRG-neurovascular unit.

circRNA landscape in dorsal root ganglia from mice with collagen antibody-induced arthritis.

Circular RNAs are a novel class of RNA molecules that are covalently closed into a ring structure. They are an epigenetic regulatory mechanism, and their best-studied function is regulation of microRNA activity. As such circular RNAs may be involved in the switch from acute to chronic pain. They have previously been studied in the context of neuropathic pain models, but their importance in inflammation-induced chronic pain models is unexplored. Microarray analysis of dorsal root ganglia collected in the late phase of collagen antibody-induced arthritis (day 59) were used to elucidate the relevance of circular RNAs in the mechanical hypersensitivity caused by this model. 120 circular RNA genes were found to be significantly differentially regulated in female BALB/c mice with collagen antibody-induced arthritis. Six genes were chosen for RT-qPCR analysis in the late (day 54-60) as well as the inflammatory (day 11-12) phase of this model. This validated an increase in circNufip1 expression in the late phase of collagen antibody-induced arthritis. Additionally, it was found that circVps13 and circMicall1 are upregulated in the inflammatory phase. Interestingly, no changes were found in dorsal root ganglia from mice injected with Freund's Complete Adjuvant (day 3) nor mice with spared nerve injury (day 20), despite their similarities to inflammatory and late phase collagen antibody-induced arthritis, respectively. This study provides evidence that mild circular RNA changes occur in dorsal root ganglia of mice with collagen antibody-induced arthritis that are, bioinformatically, predicated to be involved in processes relevant to sensitization.

DRGquant: A new modular AI-based pipeline for 3D analysis of the DRG.

BACKGROUND: The dorsal root ganglion (DRG) is structurally complex and pivotal to systems processing nociception. Whole mount analysis allows examination of intricate microarchitectural and cellular relationships of the DRG in three-dimensional (3D) space. NEW METHOD: We present DRGquant a set of tools and techniques optimized as a pipeline for automated image analysis and reconstruction of cells/structures within the DRG. We have developed an open source software pipeline that utilizes machine learning to identify substructures within the DRG and reliably classify and quantify them. RESULTS: Our methods were sufficiently sensitive to isolate, analyze, and classify individual DRG substructures including macrophages. The activation of macrophages was visualized and quantified in the DRG following intrathecal injection of lipopolysaccharide, and in a model of chemotherapy induced peripheral neuropathy. The percent volume of infiltrating macrophages was similar to a commercial source in quantification. Circulating fluorescent dextran was visualized within DRG macrophages using whole mount preparations, which enabled 3D reconstruction of the DRG and DRGquant demonstrated subcellular spatial resolution within individual macrophages. COMPARISON WITH EXISTING METHOD(S): Here we describe a reliable and efficient methodologic pipeline to prepare cleared and whole mount DRG tissue. DRGquant allows automated image analysis without tedious manual gating to reduce bias. The quantitation of DRG macrophages was superior to commercial solutions. CONCLUSIONS: Using machine learning to separate signal from noise and identify individual cells, DRGquant enabled us to isolate individual structures or areas of interest within the DRG for a more granular and fine-tuned analysis. Using these 3D techniques, we are better able to appreciate the biology of the DRG under experimental inflammatory conditions.