Sahng Y. Kim

Ocular nanoparticle toxicity and transfection of the retina and retinal pigment epithelium

Chitosan, PCEP (poly{[(cholesteryl oxocarbonylamido ethyl) methyl bis(ethylene) ammonium iodide] ethyl phosphate}), and magnetic nanoparticles (MNPs) were evaluated for the safe delivery of genes in the eye. Rabbits were injected with nanoparticles either intravitreally (IV) or subretinally (SR) and sacrificed 7 days later. Eyes were grossly evaluated for retinal pigment epithelium abnormalities, retinal degeneration, and inflammation. All eyes were cryopreserved and sectioned for analysis of toxicity and expression of either enhanced green or red fluorescent proteins. All of the nanoparticles were able to transfect cells in vitro and in vivo. IV chitosan showed inflammation in 12/13 eyes, whereas IV PCEP and IV MNPs were not inflammatory and did not induce retinal pathology. SR PCEP was nontoxic in the majority of cases but yielded poor transfection, whereas SR MNPs were nontoxic and yielded good transfection. Therefore, we conclude that the best nanoparticle evaluated in vivo was the least toxic nanoparticle tested, the MNP.

Expression of pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor (VEGF) in sickle cell retina and choroid

Pigment epithelium-derived factor (PEDF) has been shown to be an inhibitor of angiogenesis as well as a multipotent neurotrophic factor in the mammalian eye. Changes in PEDF levels have been correlated with development of retinal neovascularization in oxygen-induced retinopathy. The purpose of this study was to determine the localization and relative level of PEDF in human retinas and choroids using immunohistochemistry and evaluate the changes in PEDF and vascular endothelial growth factor (VEGF) localization and their relation to the progression of proliferative sickle cell retinopathy. Cryopreserved tissues from eyes of normal subjects and subjects with non-proliferative or proliferative sickle cell retinopathy were used with streptavidin peroxidase immunohistochemistry. A rabbit polyclonal antibody was made against recombinant human PEDF. Binding of the antibody was blocked by preincubation of the antibody with excess human recombinant PEDF. Relative levels of immunoreactivity were scored with a seven-point grading system and by microdensitometric analysis.The most prominent sites of PEDF localization in the normal eye were the vitreous condensed at the internal limiting membrane and RPE-Bruchs membrane-choriocapillaris complex. PEDF was also prominent in choroidal stroma. There was limited immunoreactivity in some cells of the neural retinas, in blood vessels and in the interphotoreceptor matrix (IPM). There was no difference in ratio (1.47 vs. 1.44) of PEDF/VEGF or the relative levels of either growth factor in the retinal vasculatures of the control subjects and perfused area of non-proliferative sickle cell retinas. The ratio was increased in the non-perfused area of the non-proliferative sickle cell retinas (2.24). In eyes with proliferative sickle cell retinopathy, elevated PEDF and VEGF immunostaining was present in viable vessels of sea fan neovascular formations as well as feeder vessels of sea fans. The PEDF/VEGF ratio in sea fans was 1.0. Immunoreactivity for PEDF was prominent in retinal vessels in non-perfused regions and in atrophic sea fans, while VEGF immunoreactivity was weak or absent in these structures. In conclusion, PEDF and VEGF were both significantly elevated in viable sea fan formations in sickle cell disease (p<0.05) but only PEDF was present in non-viable sea fans. The highest levels of PEDF in all eyes were associated with extracellular matrices (vitreous, choroidal stroma, IPM, and walls of blood vessels). PEDF might play an important role in inhibiting angiogenesis and inducing the regression of sea fans. Progression of angiogenesis may be dependent on the ratio of PEDF/VEGF.

Heat Effects on the Retina

BACKGROUND AND OBJECTIVE To study the heat and power dissipation effect of anintraocular electronic heater on the retina. The determination of thermal parameters that are nonharmful to the retina will aid in the development of an implantable intraocular electronic retinal prosthesis. MATERIALS AND METHODS In dogs, five different retinal areas were touched with a custom intraocular heater probe (1.4 x 1.4 x 1.0 mm) for 1 second while the heater dissipated 0 (control), 10, 20, 50, or 100 mW. In a second protocol, the heater was mechanically held in the vitreous cavity while dissipating 500 mW for 2 hours while monitoring intraocular temperature. The animals were observed for 4 weeks with serial fundus photography and electroretinography. The procedure was then repeated in the fellow eye. The dogs were killed and both eyes were enucleated and submitted for histology. RESULTS In experiments using protocol 1, heater settings of 50 mW or higher caused an immediate visible whitening of the retinal tissue. Histologically, this damage was evident only if the eyeswere immediately enucleated. Permanent damage was caused by heater settings of 100 mW or higher. Under protocol 2, no ophthalmologic, electroretinography, or histologic differences were noted between the groups. Temperature increases of 5 degrees C in the vitreous and 2 degrees C near the retina were noted. CONCLUSIONS The liquid environment of the eye acts as a heat sink that is capable of dissipating a significant amount of power. An electronic chip positioned away from the retina can run at considerably higher powers than a chip positioned on the retinal surface.

Mechanisms for sickle red blood cell retention in choroid.

Purpose. Although sickle (SS) red cell-mediated vaso-occlusion in retina and resultant retinopathy is well documented, the effects of SS red cells on choroidal vasculature are poorly understood. The intent of this study was to determine, using a rat model, the conditions under which retention of sickle erythrocytes in choroid occur and if that retention can be inhibited. Methods. Sickle red cells were density separated into high density (SS4) or normal density, reticulocyte-enriched fractions (SS2). Red cells were labeled with FITC and administered IV to anesthetized Sprague Dawley rats. Rats were made either hypoxic or were given TNF-a intraperitoneally 5 hours before intravenous administration of red cells. Five minutes after administration of red cells, rats were exsanguinated, the retinas removed, and choroids prepared as flat-mounts. The number of red cells retained in five high power fields of choroid was then determined. In other experiments, SS red cells were preincubated with the cyclic peptide TBC772 [inhibits binding of a4ß1 (VLA-4) and a4ß7 to their ligands], a control peptide TBC1194, or a VLA-4 neutralizing antibody before administration to the rat or antibodies against VLA-4 ligands were delivered IV before administration of SS red cells. Results. Hypoxic conditions before administration of SS red cells significantly stimulated retention of SS4 cells (P = 0.0003), but did not significantly increase retention of SS2 cells. Administration of TNF-a significantly increased retention of all types of SS red cells (P < 0.001). Preincubation of cells with anti-VLA-4 or TBC 772 inhibited retention of SS red cells in choriocapillaris of TNF-a-treated rats (P < 0.0001). Complete inhibition of cytokine-stimulated retention was also accomplished by IV administration of mono-clonal antibodies against fibronectin or its CS-1 domain, a ligand for VLA-4. Conclusions. The mechanisms for retention of SS red cells in retina and choroid appear identical: hypoxia-mediated retention of dense red cells and adherence of red cells in reticulocyte-rich fractions after cytokine stimulation. TNF-a-stimulated retention of SS red cells in choroid appears to be mediated by VLA-4, presumably on the surface of some reticulocytes. This increased retention was inhibited by a VLA-4 antagonist (TBC772), a VLA-4 neutralizing antibody or by blocking one of VLA-4s ligands, the CS-1 portion of fibronectin.