in-vivo-multimodal-imaging-analysis-mouse-laser-induced-choroidal

<em>In Vivo</em> Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this ...

Experimentica Ltd.

Loyola University Chicago

The purpose of the study was to devise a fast, reliable and sensitive cell viability assay for assessment of acute cytotoxicity on human corneal epithelial cells by using a clinically relevant exposure time. Acute cytotoxic effects of the pharmaceutical excipients benzalkonium chloride (BAC), macrogolglycerol hydroxystearate (MGHS40), polysorbate 80 (PS80) and marketed ophthalmic formulations (Lumigan(®), Monoprost(®), Taflotan(®), Travatan(®), Xalatan(®)) containing these excipients were tested. Human corneal epithelial cell (HCE-T) viability was assessed by measuring the reduction of resazurin to highly fluorescent resorufin. Expression of the tight junction proteins in HCE-T cells were characterized by immunofluorescence staining. Presence of tight junction proteins in HCE-T cells was demonstrated. BAC preserved ophthalmic formulations showed concentration-dependent and time-dependent cytotoxicity to human corneal epithelium. In contrast, no acute cytotoxicity of non-ionic stabilizing/solubilizing excipients (MGSH40 and PS80) or ophthalmic formulation containing these excipients was observed. Marketed ophthalmic formulations used for glaucoma medication show differential toxicity on human corneal epithelial cells. The present study revealed that BAC-preserved ophthalmic formulations were able to induce acute cytotoxic effects even during a clinically relevant exposure time, which was not observed with MGSH40 and PS80 excipients or ophthalmic formulations containing these excipients.

Acute cytotoxic effects of marketed ophthalmic formulations on human corneal epithelial cells.

BACKGROUND In glaucoma, non-intraocular pressure (IOP)-related risk factors can result in increased levels of extracellular glutamate, which triggers a cascade of neurodegeneration characterized by the excessive activation of N-methyl-D-aspartate (NMDA). The purpose of our study was to evaluate the glioprotective effects of memantine as a prototypic uncompetitive NMDA blocker on retinal astrocytes in the optic nerve crush (ONC) mouse model for glaucoma. MATERIAL AND METHODS Optic nerve crush was performed on all of the right eyes (n=8), whereas left eyes served as contralateral healthy controls (n=8) in Balb/c/Sca mice. Four randomly assigned mice received 2-µl intravitreal injections of memantine (1 mg/ml) after ONC in the experimental eye. One week after the experiment, optic nerves were dissec-ted and stained with methylene blue. Retinae were detached from the sclera. The tissue was immunostained. Whole-mount retinae were investigated by fluorescent microscopy. Astrocyte counts for each image were performed manually. RESULTS Histological sections of crushed optic nerves showed consistently moderate tissue damage in experimental groups. The mean number of astrocytes per image in the ONC group was significantly lower than in the healthy control group (7.13±1.5 and 10.47±1.9, respectively). Loss of astrocytes in the memantine-treated group was significantly lower (8.83±2.2) than in the ONC group. Assessment of inter-observer reliability showed excellent agreement among observations in control, ONC, and memantine groups. CONCLUSIONS The ONC is an effective method for investigation of astrocytic changes in mouse retina. Intravitreally administered memantine shows a promising glioprotective effect on mouse retinal astrocytes by preserving astrocyte count after ONC.

Simon Kaja

Experimentica Ltd.

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model