John M. Nickerson

Bile acids in treatment of ocular disease

Bear bile has been included in Asian pharmacopeias for thousands of years in treatment of several diseases, ranging from sore throat to hemorrhoids. The hydrophilic bile acids tauroursodeoxycholic acid (TUDCA) and ursodeoxycholic acid (UDCA) are the major bile acids of bear bile. Both of these are available as synthetic formulations and are approved by the health administrations of several countries for treatment of cirrhosis and gallstones. This review briefly covers the use of bear bile in Traditional Chinese Medicine, bile acid physiology, approved use of UDCA and TUDCA in Western medicine, and recent research exploring their neuroprotective properties, including in models of ocular disease.

Normal Cone Function Requires the Interphotoreceptor Retinoid Binding Protein

11-cis-retinal is the light-sensitive component in rod and cone photoreceptors, and its isomerization to all-trans retinal in the presence of light initiates the visual response. For photoreceptors to function normally, all-trans retinal must be converted back into 11-cis-retinal through a series of enzymatic steps known as the visual cycle. The interphotoreceptor retinoid-binding protein (IRBP) is a proposed retinoid transporter in the visual cycle, but rods in Irbp−/− mice have a normal visual cycle. While rods are primarily responsible for dim light vision, the ability of cones to function in constant light is essential to human vision and may be facilitated by cone-specific visual cycle pathways. We analyzed the cones in Irbp−/− mice to determine whether IRBP has a cone-specific visual cycle function. Cone electroretinogram (ERG) responses were reduced in Irbp−/− mice, but similar responses from Irbp−/− mice at all ages suggest that degeneration does not underlie cone dysfunction. Furthermore, cone densities and opsin levels in Irbp−/− mice were similar to C57BL/6 (wild-type) mice, and both cone opsins were properly localized to the cone outer segments. To test for retinoid deficiency in Irbp−/− mice, ERGs were analyzed before and after intraperitoneal injections of 9-cis-retinal. Treatment with 9-cis-retinal produced a significant recovery of the cone response in Irbp−/− mice and shows that retinoid deficiency underlies cone dysfunction. These data indicate that IRBP is essential to normal cone function and demonstrate that differences exist in the visual cycle of rods and cones.

Scleral Permeability of a Small, Single-Stranded Oligonucleotide

Developing more effective ocular drug delivery systems is essential to improving the treatment of posterior segment eye disease. The large target area provided by the sclera and potentially less vision threatening complications are advantages of transscleral administration compared to more traditional modalities of drug delivery to the posterior segment. We aimed to determine the permeability coefficient for the in vitro diffusion of a small, single-stranded, oligonucleotide across human sclera. Transscleral permeability was measured by placing 100 microL of 2.96 x 10(-4) mol single-stranded, fluorescein-labeled oligonucleotide (MW = 7998.3) on the episcleral surface of sclera mounted in a perfusion chamber. Fractions of choroidal perfusate were collected hourly for 24 hours. The permeability constant or K(trans) for the transscleral diffusion of the naked, single-stranded, fluorescein-labeled oligonucleotide was 7.67 +/- 1.8 x 10(-7) cm/s (mean +/- SEM, N = 7). The permeability constant or K(trans) after intrascleral injection of the same fluorescein-labeled oligonucleotide was 1.32 +/- 0.42 x 10(-7) (mean +/- SEM, N = 4). This analysis demonstrates that diffusion of a naked, 24-base, single-stranded, fluorescein-labeled oligonucleotide can be accomplished by both of the described methods. The ability to deliver single-stranded oligonucleotides across the sclera may prove to be advantageous given the development of several novel therapeutic strategies that use similar molecules.

Subretinal Delivery and Electroporation in Pigmented and Nonpigmented Adult Mouse Eyes

Subretinal injection offers one of the best ways to deliver many classes of drugs, reagents, cells and treatments to the photoreceptor, Müller, and retinal pigment epithelium (RPE) cells of the retina. Agents delivered to this space are placed within microns of the intended target cell, accumulating to high concentrations because there is no dilution due to transport processes or diffusion. Dilution in the interphotoreceptor space (IPS) is minimal because the IPS volume is only 10-20 μl in the human eye and less than 1 μl in the mouse eye. For gene delivery purposes, we wished to transfect the cells adjacent to the IPS in adult mouse eyes. Others transfect these cells in neonatal rats to study the development of the retina. In both neonates and adults, electroporation is found to be effective. Here we describe the optimization of electroporation conditions for RPE cells in the adult mouse eye with naked plasmids. However, both techniques, subretinal injection and electroporation, present some technical challenges that require skill on the part of the surgeon to prevent untoward damage to the eye. Here we describe methods that we have used for the past 10 years (Johnson et al. Mol Vis 14: 2211-2226, 2008).