In this study, we investigated the suitability of ultrathin and porous polyimide (PI) membrane as a carrier for subretinal transplantation of human embryonic stem cell (hESC) -derived retinal pigment epithelial (RPE) cells in rabbits. The in vivo effects of hESC-RPE cells were analyzed by subretinal suspension injection into Royal College of Surgeons (RCS) rats. Rat eyes were analyzed with electroretinography (ERG) and histology. After analyzing the surface and permeability properties of PI, subretinal PI membrane transplantations with and without hESC-RPE were performed in rabbits. The rabbits were followed for three months and eyes analyzed with fundus photography, ERG, optical coherence tomography (OCT), and histology. Animals were immunosuppressed with cyclosporine the entire follow-up time. In dystrophic RCS rats, ERG and outer nuclear layer (ONL) thickness showed some rescue after hESC-RPE injection. Cells positive for human antigen were found in clusters under the retina 41 days post-injection but not anymore after 105 days. In rabbits, OCT showed good placement of the PI. However, there was loss of pigmentation on the hESC-RPE-PI over time. In the eyes with PI alone, no obvious signs of inflammation or retinal atrophy were observed. In the presence of hESC-RPE, mononuclear cell infiltration and retinal atrophy were observed around the membranes. The porous ultrathin PI membrane was well-tolerated in the subretinal space and is a promising scaffold for RPE transplantation. However, the rejection of the transplanted cells seems to be a major problem and the given immunosuppression was insufficient for reduction of xenograft induced inflammation.

L-arginine-nitric oxide (NO) pathway participates in the physiology and in many pathological processes in the eye, such as glaucoma. The aim of the present study was to compare the ocular hypotensive effect of different NO-donors, and to get more information on the role of cyclic guanosine 3',5'-monophosphate (cGMP) in this process. The test compounds were administered topically or intravitreally in the eye of a normotensive rabbit. Intraocular pressure (IOP) was measured with a pneumatonometer after topical anesthesia. The metabolites of NO (nitrite, nitrate, NOx) and cGMP were assayed from the aqueous humor and plasma. NO-synthase (NOS) protein expression was assayed in the ciliary body by Western blotting. The maximal lowering of IOP was achieved as follows: atriopeptin III (concentration 78 (microM, decrease in IOP 50%), atriopeptin II (84 (microM 37%). 8-Br-cGMP (90 mM, 37%), zaprinast + 8-Br-cGMP (1 mM + 90 mM, 34%), L-arginine (1 mM, 29%), SNP (40 mM, 28%), nitrosocaptopril (100 mM, 28%), S-nitrosothiol (SNOG) (10 mM, 27%), YC-1 (10 (microM, 25%), zaprinast + SNP (1 mM + 40 mM, 22%), spermine NONOate (100 mM, 20%) [corrected]. The decrease in IOP lasted for 2-5 hr, except with atriopeptin II and III, when IOP values were first normalized in 6 hr and 2 days, respectively. In conclusion, the results of the present study indicate that by increasing the activity of L-arginine/NO/cGMP-pathway it is possible to lower IOP in rabbits equally to the currently used antiglaucomatous drugs.

The cytotoxicity of benzalkonium chloride (BAC) and disodium edetate (EDTA) was evaluated in vitro in rabbit corneal epithelial primary cells and in the immortalized human corneal epithelial cell line SV40. Cell injury was assessed by lactate dehydrogenase (LDH) leakage and by reduction of the tetrazolium salt WST-1 to formazan by mitochondrial metabolic activity. Cell cultures were exposed to test compounds both in serum-free and in serum-containing medium. Although WST-1 and LDH tests measured different physiological endpoints, they yielded comparable results. However, the LDH test seemed less reliable due to great variation. The use of serum was found to result in lower toxicity of the compounds in both tests. The rabbit primary cell culture and the human corneal cell line were quite similar in their responses to BAC and EDTA. The human cell line is a promising in vitro alternative in oculotoxicity testing.

Background:Apoptosis is a an important process in corneal development, homeostasis, and disease. This study was performed to determine for the first time basic temporal apoptotic features of SV-40 immortalized human corneal epithelial (HCE) cells. Additionally, we introduce a sensitive analysis of confocal microscopic images to measure the kinetics of staurosporine (STS) induced phosphatidylserine (PS) membrane translocation and early nuclear morphological changes. Methods: HCE cells were cultured in the presence of STS to induce apoptosis. Caspase-3 activity was measured with the fluorogenic substrate z-DEVD-rhodamine 110. We determined mitochondrial viability with a 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzenedisulfonate reduction assay, and chromatin degradation with a fluorometric method using 4,6-diamidino-2-phenylindole (DAPI). Membrane translocation of PS and nuclear alterations were assessed by quantitative fluorescence microscopy. Image processing routines were written in interactive data language (IDL).

This study was undertaken to investigate the use of the in vitro test WST-1, an assay of cell proliferation and viability, for a preliminary safety evaluation of topical ophthalmic preparations. The cytotoxicity of two surfactants, benzalkonium chloride (BAC) and polyoxyethylene-20-stearyl ether (Brij78, PSE) was independently investigated in four laboratories in the EU by using an immortalized human corneal epithelial (HCE) cell line. The HCE cells were exposed to BAC and PSE for 5 min, 15 min, and 1 hour, and the results of the HCE-WST-1 tests were collected and compared. After one-hour exposure, the EC(50) values in BAC-treated cells in the presence of serum ranged between 0.0650 +/- 0.0284 (mean +/- SD) mM, and those in the absence of serum 0.0296 +/- 0.0081 mM. The corresponding values for PSE were 0.0581 +/-.0300 mM and 0.0228 +/-.0063 mM. There were variations in the results between different laboratories, with coefficients of variation ranging from 31 to 121%, mean 58%. The use of one-hour exposure time is to be preferred, and the elimination of serum in the culture medium is recommended to avoid both underestimation of toxic effects and variability of the test results.