Kobe University develops new technique for high-resolution observation of retina in living diabetic mice

A research group led by Associate Professor Yoshihisa Tachibana, Graduate Student Noriyuki Sotani, Lecturer Sentaro Kusuhara, and Professor Makoto Nakamura from the Graduate School of Medicine at Kobe University announced on October 7 that they have developed a new technique enabling high-resolution observation of the mouse retina while alive. Using two-photon microscopy, they successfully captured in real-time the excessive activity of retinal microglia in diabetic retinopathy, which could not be detected with conventional techniques. In the future, this is expected to be implemented in clinical settings as a noninvasive diagnostic method for the human retina. The results were published in PNAS on October 8.

The retina is composed of neurons that receive light, glial cells that support them, cells that constitute blood vessels, and other components. These cells maintain neural activity and regulate blood flow through a mechanism called the "neurovascular unit," preserving normal visual function. When the balance of neural activity maintenance and blood flow regulation is disrupted, diseases such as diabetic retinopathy progress. Diabetic retinopathy is one of the three major complications of diabetes and ranks among the leading causes of blindness.

For a long time, it was believed that vision loss occurred due to vascular damage, but in recent years it has been found that abnormalities in nerves and immune cells begin first. In particular, the role of microglia, immune cells resident in the retina, has attracted attention. Microglia constantly move their processes while monitoring their surroundings and initiate inflammatory responses when abnormalities occur.

On the other hand, the dynamics of microglia were not well understood because observation in the living state was difficult.

In this study, the researchers developed a method to observe the retinas of living mice with high resolution and ease using two-photon microscopy.

By combining a device for head fixation, a custom-made contact lens to protect the cornea, and a special objective lens capable of clearly observing deep into the retina, they were able to clearly observe the retina in living animals. They achieved clear observation without using the expensive and complex "adaptive optics" that were previously necessary.

When they examined the retinas of mice with drug-induced diabetes using the new observation method, abnormalities in microglial processes that were previously unknown became apparent.

In the retinas of healthy mice, microglia move their processes slowly. In diabetic mice, the movement of processes became vigorous, and surveillance activity was confirmed to be excessively enhanced.

Furthermore, when liraglutide, a diabetes treatment drug, was administered to diabetic mice, the excessively active movement of microglial processes was calmed, and it was confirmed that activity levels returned to levels comparable to healthy mice. Since this improvement effect appeared regardless of blood glucose reduction, it was suggested that liraglutide may have an action that suppresses abnormalities in microglia, which are immune cells of the retina. There is a possibility that the disease could be detected at an early stage by using excessive microglial activity as an indicator.

Tachibana commented: "Two-photon microscopy is a device that can observe living tissue with minimal invasiveness. It has conventionally been used to observe the cerebral cortex, but observation of cells and blood vessels in the retina deep inside the eyeball was difficult. In this study, through trial and error with our graduate student, we have made it possible to easily observe the retina. The retina can be called a 'window' to the central nervous system, and it is hoped that this will lead not only to the diagnosis of retinal diseases but also to the elucidation of diagnostic and pathological mechanisms of central nervous system diseases."

Journal Information
Publication: PNAS
Title: Transpupillary in vivo two-photon imaging reveals enhanced surveillance of retinal microglia in diabetic mice
DOI: 10.1073/pnas.2426241122