Corneal assay

The corneal assay consists in positioning an angiogenesis inducer (tumor tissue, cell suspension, growth factor) into a pocket made in the cornea in order to evoke vessel outgrowth from the peripherally located limbal vasculature. This assay, with respect to the other in vivo assays, has the advantage of measuring only new blood ves-... 

Opacity. Corneal opacity is the most heavily weighted of the components of the Draize eye score (80 out of 110 possible points) (Conquet et al., 1977). Thus, an in vitro system that provides an accurate measure of opacification should contribute substantially toward in vitro modeling of the classical Draize test. Two assays attempting to model this process are discussed. 

Gautheron, P.D. and Sina, J.F. (1990). The bovine corneal opacity, an in vitro assay of ocular irritancy. Toxicologist 10 258. 

It is well recognized that in vitro angiogenesis assays can have clear advantages. However, the major drawback of all of these assays is that they require the endothelial cells to be removed from their natural microenvironment, which alters their physiological properties. To study angiogenesis in vivo, the most frequently used assay systems exploit chicken chorio-allanto-ic membrane (CAM) [28,60], the corneal pocket [61], transparent chamber preparations such as the dorsal skin fold chamber [62,63], the cheek pouch window [64] and polymer matrix implants [65,66]. 

The corneal pocket assay and the window preparations are designed to measure vessel formation after addition of stimulators. These assays can for instance be used for the study of angiogenic potential of human tumours. These models are also suitable for pre-clinical testing of angiogenesis inhibitors. 

Another effect of LPS may be the inhibition of tumor angiogenesis. This was evidenced in melanoma B16- bearing C57BL/6 mice treated i.v. with the lipid A GL-60, after an IFN-y injection [114], It was confirmed in the same model using DT-5461 alone injected i.v. [94]. This effect of LPS is likely to be due to the production of TNF-a. This cytokine may be toxic for the endothelium [64], inhibiting at the same time the motility and proliferation of endothelial cells [59]. Sato et al. [115] showed that TNF-a inhibits tumor-induced angiogenesis in a rabbit corneal pocket assay. On the contrary, Vukanovic and Isaacs [116] concluded that TNF-... 

The growth rate of mammalian cells in the presence of Cell-Tak adhesive was assayed to evaluate any potential adverse effects caused by this protein. Bovine corneal endothelial cell (BCE) stocks were grown to confluency in MEM plus 15% calf serum, trypsinized, and washed several times by centrifugation on MEM. Suspensions (5 x 104 cells/mL) were seeded onto untreated 35-mm dishes (control) and dishes with Cell-Tak protein (5 fig/cm2) in MEM with 15% calf serum. At various time points during the incubation at 37 °C with 5% CO2, triplicate plates were removed. The attached cells were then trypsinized from the surface, washed, and counted in a hemacytometer. 

Fig. 9.1. The corneal micropocket assay a micropocket is surgically produced in the comeal stroma by a pliable spatula (a) and the sample is inserted in the micropocket (b). The newly formed vessels protrade from the limbal vasculature (c, t=0) and progress toward the implanted stimulus (d, t = 7 days).

Sina, J.F. Validation of the bovine corneal opacity-permeability assay as a predictor of ocular irritation potential. In Vitro Toxicol. 1994, 7 (3), 283-290. 

As described by Gautheron P, Dukic M, Alix D, and Sina JF (1992) Bovine corneal opacity and permeability test An in vitro assay of ocular irritancy. Fundamentals of Applied Toxicology 18(3) 442-449. 

None of the in vitro alternative eye tests has proven applicable as a valid replacement for the Draize eye irritation test or has been acceptable for regulatory purposes (Table 5), though some are considered either reliable or reproducible. The most frequently used test has been the ex vivo bovine cornea opacity and permeability assay. The newer human corneal equivalents system, an in vitro culture of immortalized human corneal cells that develops into... 

Bovine corneal cup method inflammatory response releasing chemotactic factors and then reacted with neutrophils Bovine corneal cup assay inflammatory response releasing specific mediators (histamine, serotonin, prostaglandins, leukotrienes, thromboxanes) that can be collected in the bath medium and quantitated by chemical assay Rat vaginal tissue assay similar to bovine corneal cup assay with release of specific mediators Fertile chicken egg chorioallantoic membrane (CAM) assay scoring for vascular changes in the membrane blood vessels with fluorescein dye as well necrotic damage... 

The Cytosensor Microphysiometer (CM) test method is currently under discussion (draft test guideline on the Cytosensor Microphysiometer) at OECD level [34], It is a cytotoxicity and cell-function based in vitro assay that is performed on a sub-confluent monolayer of adherent mouse L929 fibroblasts cultured in a sensor chamber using a pH-meter to detect changes in acidity [35]. The CM test method serves as an in vitro model system for the cytotoxic action of a test chemical on the cell membranes of the corneal and conjunctival epithelium where the irritant chemical would be... 

The lack of full reversibility within 21 days of application is a critical parameter to identify substances inducing serious eye damage. If the majority of the models currently accepted from a regulatory point of view (see Section 3) may allow to predict such effects, they were not specifically designed and/or do not specifically address the reversibility/persistence of effects. This may be particularly of relevance depending upon the types of materials to be evaluated, and if in vivo irreversible effects are expected. As such, a number of in vitro test methods have been developed and optimized to distinguish persistent from reversible ocular effects. These include the use of histopathology and Depth of Injury of effects (Dol), the Porcine Corneal Ocular Reversibility Assay (PorCORA) and the Ex-Vivo Eye Irritation test (EVEIT). 

The Porcine Corneal Ocular Reversibility Assay (PorCORA)... 

Porcine corneal models have been used to develop in vitro/ex vivo models able to detect recovery of ocular injury. In preliminary studies porcine corneas cultured for at least 120 h showed regeneration of the damaged stratified epithelium by treatment with 3 % SLS and Ethanol [84], The model was further optimized and developed towards an ocular irritancy assay based on porcine corneas with reversibility as an endpoint, called the Porcine Corneal Ocular Reversibility Assay (PorCORA) [85]. The assay uses an air-interface porcine corneal culture system, and is maintained in culture for 21 days, similar to the in vivo observation period, to determine reversibility of corneal injury as measured by sodium fluorescein and to detect potential compromised epithelial barrier function. 

Additional in vitro models and assays for eye irritation testing have been proposed in the past. An example is the porcine-based corneal opacity and permeability (PCOP) assay to predict eye irritation of water-soluble cosmetic ingredients [99]. Porcine corneas were chosen due to their advantages in comparison to the bovine corneas such as no concerns about encephalopathy diseases, regular supply of eyes with an acceptable level of quality from a slaughterhouse, more closely related to the human cornea, and their use in ophthalmic research. Using a modified holder and adapted experimental procedures 50 cosmetic ingredients were tested and compared to the in vivo Draize MAS scores. Based on their results, the study concluded that the PCOP could better predict the irritation classes than the BCOP assay. 

Maurer JK, Parker RD, Jester JV (2002) Extent of initial corneal injury as the mechanistic basis for ocular irritation key findings and recommendations for the development of alternative assays. Regul Toxicol Pharmacol 36 106-117... 

Cooper KJ, Earl LK, Harbell J, Raabe H (2001) Prediction of ocular irritancy of prototype shampoo formulations by the isolated rabbit eye (IRE) test and bovine corneal opacity and permeability (BCOP) assay. Toxicology In Vitro 15 95-103... 

Raabe H, Bruner L, Snyder T, Wilt N, Harbell J (2005) Optimisation of an in vitro long term corneal culture assay. Poster presented at the 5th World Congress on alternatives and animal use in the life sciences, Berlin, Germany... 

Piehl M, Gilotti A, Donovan A, DeGeorge G, Cerven D (2010) Novel cultured porcine corneal irritancy assay with reversibility endpoint. Toxicol In Vitro 24 231-239... 

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