Three-dimensional Mapping of the Retinal Neurovascular Unit in Health and Diabetes

Abstract

Diabetic Retinopathy (DR), a leading cause of adult blindness, is a progressive development of microvascular and neurovascular damage in the retina which leads to vision-threatening complications. The disruption of the neurovascular unit (NVU) is a primary factor in the pathogenesis of DR. The NVU is an interdependent unit made up of many cell types including pericytes, endothelium, glial cells and neurons, which work synergistically to maintain the function of the retina and allows adaptation to change in the physiological environment; for example, by regulating blood flow and by maintaining the blood retinal barrier (BRB). To assist with improving our understanding of retinal biology, and developing strategies to correct NVU dysfunction, detailed knowledge of the heterocellular interactions of the NVU is required. To address this issue, I have used serial block face scanning electron microscopy (SBF-SEM), and computational image reconstruction, which has enabled the first three-dimensional ultrastructural analysis of the NVU in retinal capillaries. Examination of the data in the x-, y- and z-planes was performed with the use of semi-automated computational image analysis tools including segmentation, 3D image reconstruction and quantitation of cell proximities to provide visualisation and analysis of the data. Heterocellular relationships within the retina were assessed in mouse and human tissues in health and diabetes. Prominent features of the capillary arrangements in 3D were the extensive sheath like coverage by singular pericytes. They appeared in close register to the basement membrane (BM) with which they interwove in a complex mesh-like appearance. Breaks in the basement membrane appeared to facilitate pericyte interactions with other NVU cell types. There were frequent, close (<10 nm) pericyte to endothelial interactions with direct contact points and peg-and-socket-like morphology, usually appearing 2-4 times per micron with each formation spanning across several sections. Macroglia typically intervened between neurons and capillary structures; however, regions were identified where neurons came into closer contact with the BM. With the onset of diabetes, endothelial cells and pericytes showed areas of detachment from the BM, leaving intermittent gaps between their plasma membranes and the BM. These separations spanned a minimum depth of 200 nm in the z-dimension and exhibited a minimum width of 20 nm. Pericyte-endothelial cell interactions via peg and socket formations in non-diabetic capillaries show both cell membranes in close apposition, however, there appeared to be space surrounding the peg in the socket area of diabetic capillaries. Similar electron lucent gaps were present in the endothelium of diabetic capillaries. Moreover, showed electron-lucent tubules traversing its structure. These tubules, appearing as white, electron-lucent holes, varied in size and did not open on either side, suggesting they do not form conventional channels within the endothelium. An increase in the number of leukocytes were present in the luminal space of diabetic capillaries, which were found to make direct communication with endothelial projections. This work provides new information on the ultrastructural changes in the murine retinal NVU during the onset and progression of diabetes, which in turn can serve as a platform to inform future studies aimed at delaying or preventing the progression of retinopathy.