In vitro effects of insulin and RPE on choroidal and scleral components of eye growth in chicks.

In chick eyes, exogenous insulin prevents the choroidal thickening caused by wearing positive lenses and increases ocular elongation and scleral glycosaminoglycan (GAG) synthesis, an indicator of eye growth. Using in vitro eye-cups, a novel experimental system, we examined the role of the retinal pigment epithelium (RPE) and insulin on choroidal thickness and scleral GAG synthesis. Specifically, we asked whether insulin causes the release of diffusible factors from the RPE that affect the choroid. We studied the effect of insulin on choroidal thickness and scleral GAG synthesis by making eye-cups consisting of RPE, choroid, and sclera (RCS), choroid and sclera (CS), or just sclera from pairs of eyes. One eye-cup was cultured in 0.037, 0.37, 3.7 or 37 μM insulin dissolved in L-15 medium, and its pair was cultured in L-15 medium without insulin. Choroidal thickness in eye-cups was measured by A-scan ultrasonography before and after 20 h of incubation. Sulfate incorporation into GAGs (scleral GAG synthesis) was measured after 44 h of incubation. To further study the effect of RPE and insulin on the choroids, we prepared pairs of CS eye-cups cultured with vs. without RPE transplanted from donor eyes, in the presence or absence of 37 μM insulin. To study if insulin caused the RPE to produce diffusible factors that affected the choroid, we prepared medium conditioned by the RPE in the presence (experimental conditioned medium) or absence (control conditioned medium) of 37 μM insulin for 20 h. Experimental and control conditioned media were pooled separately, and an equal volume of medium containing 37 μM insulin was added to both experimental and control media. Pairs of CS eye-cups were cultured in conditioned medium (experimental vs. control). Choroidal thickness was measured before and after 20 h of incubation. Choroids in all eye-cups thickened after 20 h of incubation. Insulin reduced this natural choroidal thickening seen in culture significantly, but only if the RPE was present. This effect was dose-dependent and strongest at 37 μM. Insulin increased scleral GAG synthesis in both RCS and CS eye-cups, having a greater effect in the CS eye-cups. Insulin had no effect on scleral GAG synthesis in scleral eye-cups. Choroids of CS eye-cups cultured with transplanted RPE plus insulin thickened significantly less than choroids of eye-cups cultured with insulin but without the RPE. The reduction in choroidal thickening was similar to that seen in eye-cups with intact RPE (RCS). Choroidal thickening of CS eye-cups cultured with experimental conditioned medium was significantly reduced compared with their pairs cultured with control conditioned medium. In vitro, as in vivo, insulin prevents choroidal thickening and increases scleral GAG synthesis. Insulin causes the RPE to synthesize diffusible molecules that inhibit choroidal thickening. Insulin might also cause the choroid to produce secondary signals that affect scleral GAG synthesis.