PurposeLoss of retinal capillary endothelial cells and pericytes through apoptosis is an early event in diabetic retinopathy (DR). Inflammatory pathways play a role in early DR, yet the biochemical mechanisms are poorly understood. In this study, we investigated the role of indoleamine 2,3-dioxygenase (IDO), an inflammatory cytokine-inducible enzyme, on retinal endothelial apoptosis and capillary degeneration in the diabetic retina.MethodsIDO was detected in human and mouse retinas by immunohistochemistry or Western blotting. Interferon-γ (IFN-γ) levels were measured by ELISA. IDO levels were measured in human retinal capillary endothelial cells (HREC) cultured in the presence of IFN-γ ± 25 mM D-glucose. Reactive oxygen species (ROS) were measured using CM-H2DCFDA dye and apoptosis was measured by cleaved caspase-3. The role of IDO in DR was determined in IDO knockout (IDO−/−) mice with streptozotocin-induced diabetes.ResultsThe IDO and IFN-γ levels were higher in human diabetic retinas with retinopathy relative to nondiabetic retinas. Immunohistochemical data showed that IDO is present in capillary endothelial cells. IFN-γ upregulated the IDO and ROS levels in HREC. The blockade of either IDO or kynurenine monooxygenase led to inhibition of ROS in HREC. Apoptosis through this pathway was inhibited by an ROS scavenger, TEMPOL. Capillary degeneration was significantly reduced in diabetic IDO−/− mice compared to diabetic wild-type mice.ConclusionsThe results suggest that the kynurenine pathway plays an important role in the inflammatory damage in the diabetic retina and could be a new therapeutic target for the treatment of DR.

In the healthy ageing Chinese eye, IOP remains unchanged, while Fa slows, which is counterbalanced by slowing of Fu. Aqueous humour exits the eye preferentially through the trabecular route at all ages. Ageing is also associated with shallowing of the anterior chamber and thinning of the cornea. A slower Fa with lower outflow facility supports existence of autoregulatory mechanisms.

This protocol provides a step-by-step guide to shotgun sphingolipid analysis of aqueous humor. We describe the Bligh and Dyer crude lipid extraction method and electrospray ionization tandem mass spectrometry (ESI-MS/MS) coupled with MZmine 2.21 data processing for identification and ratiometric quantitation of sphingosine, sphingosine-1-phosphate, sphingomyelin, and ceramide.