Purposes: First, to develop an improved retinal capillary endothelial cell culture system which exhibits some of the physiologic features of the bloodretinal barrier; second, to use this model to determine how histamine and chemical conditions of diabetes effect expression of the tight junction protein, ZO-1; and third, to discuss application of the Henle-Koch postulates to the problem of diabetic retinopathy.
Methods: Bovine retinal capillary endothelial cells were exposed to varying serum and growth factor concentrations, as well as astrocyte-conditioned medium, in order to establish a model of the blood-retinal barrier. Cells were also exposed to varying concentrations of histamine, and of insulin and glucose. The expression of ZO-1 tight junction protein was determined by immunocytochemistry and immunoblotting.
Results: Modified concentrations of growth factors reduced endothelial cell proliferation, without reducing viability. Astrocyte conditioned medium increased ZO-1 protein content. Histamine reduced ZO-1 protein content. Both high glucose (20mM) and low insulin (10(-12)M) reduced ZO-1 protein content compared to control conditions (5mM glucose and 10(-9) M insulin).
Conclusions: Control of culture conditions results in a more physiologic in vitro model of the blood-retinal barrier. Soluble factors from astrocytes promote tight junction formation. Both histamine and chemical conditions of diabetes diminish tight junction formation. These factors may mediate blood-retinal barrier breakdown in diabetic retinopathy. Henle-Koch postulates for diabetic retinopathy are presented.