Thalidomide is a potent teratogen causing dysmelia (stunted limb growth) in humans. We have demonstrated that orally administered thalidomide is an inhibitor of aniogenesis induced by basic fibroblast growth factor in a rabbit cornea micropocket assay. Experiments including the analysis of thalidomide analogs revealed that the antaniogenic activity correlated with the teratogenicity but not with the sedative or the mild immunosuppressive properties of thalidomide. Electron microscopic examination of the corneal neovascularization ofthafidomide-treated rabbits revealed specific ultrastructural changes similar to those seen in the deformed limb bud vasculature of thalidomide-treated embryos. These experiments shed light on the mechanism of thalidomide's teratogenicity and hold promise for the potential use of thalidomide as an orally adminsered drug for the treatment of many diverse dease dependent on angiogenesis.Thalidomide is a potent teratogen. It was developed by Chemie Grunenthal in the 1950s as a sedative that appeared so nontoxic in rodent models that a LD50 could not be established. In 1961, McBride (1) and Lenz (2) described the association between limb defects in babies and maternal thalidomide usage. Although humans are exquisitely sensitive to the teratogenic effects of thalidomide, experiments in rodents failed to reveal similar effects (3, 4). Teratogenic effects could be experimentally reproduced by the administration of thalidomide to pregnant rabbits at an oral dose of 100-300 mg per kg per day (5, 6). Over the past 30 years, the mechanism of thalidomide's teratogenicity has been extensively studied but has remained unsolved (7).We now postulate that the limb defects seen with thalidomide were secondary to an inhibition of blood vessel growth in the developing fetal limb bud. The limb bud is unique in requiring a complex interaction of both angiogenesis and vasculogenesis during development (8). Vasculogenesis is the formation of a capillary bed from endothelial cells that have differentiated from mesenchymal precursors. Angiogenesis is the formation of new blood vessels from sprouts ofpreexisting vessels. Therefore, the limb bud would be a particularly vulnerable target to a teratogen that inhibited endothelial cell function. We chose to examine the effect of thalidomide on growing vasculature in the chicken chorioallantoic membrane and in the rabbit cornea. MATERIALS AND METHODSChicken chorioallantoic membrane (CAM) assays were performed as described (9, 10) and the effects on the developing vasculature were recorded at 48 h after implantation of the 0.5% carboxymethylcellulose pellet containing various drugs. Corneal

These data show that protecting retinal neurons from diabetic stress also reduces VEGF expression and release, while inhibiting VEGF leads to exacerbation of apoptosis. These observations suggest that the retina in early DR releases VEGF as a prosurvival factor. Neuroprotective agents may decrease the need of VEGF production by the retina, therefore limiting the risk, in the long term, of pathologic angiogenesis.

Neuronal injury plays a major role in diabetic retinopathy (DR). Our hypothesis was that the balance between neuronal death and survival may depend on a similar equilibrium between apoptosis and autophagy and that a neuroprotectant may act by influencing this equilibrium. Ex vivo mouse retinal explants were treated with high glucose (HG) for 10days and the somatostatin analog octreotide (OCT) was used as a neuroprotectant. Chloroquine (CQ) was used as an autophagy inhibitor. Apoptotic and autophagic markers were evaluated using western blot and immunohistochemistry. HG-treated explants displayed a significant increase of apoptosis paralleled by a significant decrease of the autophagic flux, which was likely to be due to increased activity of the autophagy regulator mTOR (mammalian target of rapamycin). Treatment with OCT rescued HG-treated retinal explants from apoptosis and determined an increase of autophagic activity with concomitant mTOR inhibition. Blocking the autophagic flux with CQ completely abolished the anti-apoptotic effect of OCT. Immunohistochemical observations showed that OCT-induced autophagy is localized to populations of bipolar and amacrine cells and to ganglion cells. These observations revealed the antithetic role of apoptosis and autophagy, highlighting their equilibrium from which neuronal survival is likely to depend. These data suggest the crucial role covered by autophagy, which could be considered as a molecular target for DR neuroprotective treatment strategies.

There is indication that nutritional supplements protect retinal cells from degeneration. In a previous study, we demonstrated that dietary supplementation with an association of forskolin, homotaurine, spearmint extract and B vitamins efficiently counteracts retinal dysfunction associated with retinal ganglion cell (RGC) death caused by optic nerve crush. We extended our investigation on the efficacy of dietary supplementation with the use of a mouse model in which RGC degeneration depends as closely as possible on intraocular pressure (IOP) elevation. In this model, injecting the anterior chamber of the eye with methylcellulose (MCE) causes IOP elevation leading to RGC dysfunction. The MCE model was characterized in terms of IOP elevation, retinal dysfunction as determined by electrophysiological recordings, RGC loss as determined by brain-specific homeobox/POU domain protein 3A immunoreactivity and dysregulated levels of inflammatory and apoptotic markers. Except for IOP elevation, dysfunctional retinal parameters were all recovered by dietary supplementation indicating the involvement of non-IOP-related neuroprotective mechanisms of action. Our hypothesis is that the diet supplement may be used to counteract the inflammatory processes triggered by glial cell activation, thus leading to spared RGC loss and the preservation of visual dysfunction. In this respect, the present compound may be viewed as a potential remedy to be added to the currently approved drug therapies for improving RGC protection.

Flickering light increases metabolic demand in the inner retina. Flicker may exacerbate defective mitochondrial function in glaucoma, which will be reflected in the pattern electroretinogram (PERG), a sensitive test of retinal ganglion cell (RGC) function. We tested whether flicker altered the PERG of DBA/2J (D2) glaucomatous mice and whether vitamin B3-rich diet contributed to the flicker effect. D2 mice fed with either standard chow (control, n = 10) or chow/water enriched with nicotinamide (NAM, 2000 mg/kg per day) (treated, n = 10) were monitored from 3 to 12 months. The PERG was recorded with superimposed flicker (F-PERG) at either 101 Hz (baseline) or 11 Hz (test), and baseline-test amplitude difference (adaptation) evaluated. At endpoint, flat-mounted retinas were immunostained (RBPMS and mito-tracker). F-PERG adaptation was 41% in 3-month-old D2 and decreased with age more in control D2 than in NAM-fed D2 (GEE, p < 0.01). At the endpoint, F-PERG adaptation was 0% in control D2 and 17.5% in NAM-fed D2, together with higher RGC density (2.4×), larger RGC soma size (2×), and greater intensity of mitochondrial staining (3.75×). F-PERG adaptation may provide a non-invasive tool to assess RGC autoregulation in response to increased metabolic demand and test the effect of dietary/pharmacological treatments on optic nerve disorders.