Isolation of Mouse Retinal Capillaries and Subendothelial Matrix for Stiffness Measurement Using Atomic Force Microscopy

Retinal capillary degeneration is a clinical hallmark of the early stages of diabetic retinopathy (DR). Our recent studies have revealed that diabetes-induced retinal capillary stiffening plays a crucial and previously unrecognized causal role in inflammation-mediated degeneration of retinal capillaries. The increase in retinal capillary stiffness results from the overexpression of lysyl oxidase, an enzyme that crosslinks and stiffens the subendothelial matrix. Since tackling DR at the early stage is expected to prevent or slow down DR progression and associated vision loss, subendothelial matrix, and capillary stiffness represent relevant and novel therapeutic targets for early DR management. Further, direct measurement of retinal capillary stiffness can serve as a crucial preclinical validation step for the development of new imaging techniques for non-invasive assessment of retinal capillary stiffness in animal and human subjects. With this view in mind, we here provide a detailed protocol for the isolation and stiffness measurement of mouse retinal capillaries and subendothelial matrix using atomic force microscopy.