Abstract
Purpose :
We now know that there are at least 3 different classes of macrophages in the retina and CNS. In health, microglia and perivascular macrophages (PVM) are the only classes present, whereas monocyte-derived macrophages also infiltrate in the disease context. Discerning these populations is technically challenging given their overlapping phenotypic marker expression using conventional methods (e.g., IBA-1, CD45, CX3CR1, CD11b). Deciphering these populations is now possible with new genetic lineage tracing methods in mouse models. The current study is focused on characterizing PVMs in steady state because these cells could be critical in trafficking molecules and immune cells across the blood retina barrier due to PVMs unique spatial positioning at the interface of the vasculature and neural parenchyma.
Methods :
We analyzed our published single-cell RNA sequencing (scRNA-seq) dataset (PMID: 30850344) to identify differentially expressed genes in PVMs versus microglia, and trace monocyte-derived cells, in steady state. Once genes of interest were identified, we then leveraged conditional genetic labeling to validate and characterize PVMs using confocal microscopy. In addition, we also incorporated immunolabeling of other markers defined as differentially expressed by PVM, which included CD206, LYVE1, P2RY12, and IBA-1. Isolectin B4 (IB4) was used to visualize the vasculature.
Results :
Our scRNA-seq dataset inferred that PVMs highly express Pf4, Mrc1, and Lyve1 the retina. Based on these data, we bred Pf4-Cre;R26-iDTR mice (Diphtheria toxin receptor). Using immunolabelling with anti-DTR, we found PVMs located perivascularly, as expected. Moreover, in vivo pulse DT administration induced conditional depletion of these PVMs. In terms of PVM distribution, we found that these cells were enriched near the optic nerve head and were also sporadically distributed throughout the retinal periphery. We also found that CD206 marks the majority of PVM, whereas LYVE1 was expressed in only a subset of these cells.
Conclusions :
Our research indicates that PVMs are primarily enriched around retinal vessels near the optic nerve head. Pf4 is a gene expressed in retinal PVM, possibly serving as a specific marker for these cells. Furthermore, our model can deplete PVMs through the administration of DT, enabling future detailed studies of PVM function and their association with diseases.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.